Induction Of EAE Research Articles

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles attenuate experimental autoimmune encephalomyelitis via regulating pro and anti-inflammatory cytokines

The therapeutic effects of mesenchymal stem cells-extracellular vesicles have been proved in many inflammatory animal models. In the current study, we aimed to investigate the effect of extracellular vesicles (EVs) derived from human umbilical cord-MSC (hUCSC-EV) on the clinical score and inflammatory/anti-inflammatory cytokines on the EAE mouse model. After induction of EAE in C57Bl/6 mice, they were treated intravenously with hUCSC-EV or vehicle. The clinical score and body weight of all mice was registered every day. On day 30, mice were sacrificed and splenocytes were isolated for cytokine assay by ELISA. Cytokine expression of pro-/anti-inflammatory cytokine by real-time PCR, leukocyte infiltration by hematoxylin and eosin (H&E) staining, and the percent of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) positive cells by immunohistochemistry were assessed in the spinal cord. Our results showed that hUCSC-EV-treated mice have lower maximum mean clinical score (MMCS), pro-inflammatory cytokines, and inflammatory score in comparison to the control mice. We also showed that hUCSC-EV administration significantly improved body weight and increased the anti-inflammatory cytokines and the frequency of Treg cells in the spleen. There was no significant difference in the percent of GFAP and MBP positive cells in the spinal cord of experimental groups. Finally, we suggest that intravenous administration of hUCSC-EV alleviate induce-EAE by reducing the pro-inflammatory cytokines, such as IL-17a, TNF-α, and IFN-γ, and increasing the anti-inflammatory cytokines, IL-4 and IL-10, and also decrease the leukocyte infiltration in a model of MS. It seems that EVs from hUC-MSCs have the same therapeutic effects similar to EVs from other sources of MSCs, such as adipose or bone marrow MSCs.

Developmental ablation of mature oligodendrocytes exacerbates adult CNS demyelination

Multiple sclerosis (MS) is a CNS neurodegenerative autoimmune disease characterized by loss of oligodendrocytes and myelin in the brain and the spinal cord that results in localized functional deficits. Several risk factors have been associated with MS, however none fully explain the enhanced susceptibility seen in older individuals. Epidemiological data, based on geographical prevalence studies suggest that susceptibility is established early in life and frequently long before the diagnosis of disease raising the possibility that developmental events influence adult disease onset and progression. Here we test the hypothesis that selective loss of mature oligodendrocytes during postnatal development results in enhanced susceptibility to a demyelinating insult to the mature CNS. A transgenic mouse model was utilized to specifically induce apoptotic cell death in a subset of mature oligodendrocytes (MBP-iCP9) during the first 2 postnatal weeks followed by either a local LPC spinal cord injection or the induction of EAE in the adult animal. Immunostaining, immunoblotting, behavioral testing, and electron microscopy were utilized to examine the differences in the response between animals with developmental loss of oligodendrocytes and controls. We show that during development, oligodendrocyte apoptosis results in transient reductions in myelination and functional deficits that recover after 10–14 days. Compared to animals in which oligodendrocyte development was unperturbed, animals subjected to postnatal oligodendrocyte loss showed delayed recovery from an LPC lesion to the mature spinal cord. Unexpectedly, the induction and severity of MOG induced EAE was not significantly altered in animals following oligodendrocyte developmental loss even though there was a substantial increase in spinal cord tissue damage and CNS inflammation. It is unclear why the elevated glial responses seen in developmentally compromised animals were not reflected in enhanced functional deficits. These observations suggest that developmental loss of oligodendrocytes results in long lasting tissue changes that alter its response to subsequent insults and the capacity for repair in the adult.

Developmental loss of oligodendrocytes hinders adult CNS remyelination and increases astroglial and microglial activation

Multiple sclerosis, a neurodegenerative autoimmune disease characterized by loss of oligodendrocytes and myelin in the brain and spinal cord, results in localized functional deficits. Several risk factors have been associated with MS, however none can fully explain enhanced susceptibility to MS. Epidemiological data, based on geographical prevalence studies suggest susceptibility is established early in life and frequently long before disease diagnosis indicating developmental events influence adult disease progression. Here we test the hypothesis that loss of mature oligodendrocytes during postnatal development results in enhanced susceptibility to a second demyelinating insult. A transgenic mouse model was utilized to specifically induce apoptosis in a subset of mature oligodendrocytes (MBP‐iCP9) during the first 2 postnatal weeks followed by either a local LPC spinal cord injection or the induction of EAE in the adult. Immunostaining, immunoblotting, behavioral testing, and scanning electron microscopy were utilized to examine the differences between groups. We show that during development, oligodendrocyte apoptosis results in transient reductions in myelination and functional deficits that recover after 10–14 days. Compared to animals in which oligodendrocyte development was unperturbed, animals subjected to postnatal oligodendrocyte ablation showed delayed recovery from an LPC lesion. Unexpectedly, the induction and severity of EAE was not significantly altered in animals following oligodendrocyte ablation even though there was a substantial increase in spinal cord tissue damage and CNS inflammation. It is currently unclear why these changes are not reflected in enhanced functional deficits. These observations suggest that developmental loss of oligodendrocytes results in long lasting tissue changes that alter its capacity for repair in the adult.Support or Funding InformationNIH834857

The macrophage migration inhibitory factor pathway in human B cells is tightly controlled and dysregulated in multiple sclerosis.

In MS, B cells survive peripheral tolerance checkpoints to mediate local inflammation, but the underlying molecular mechanisms are relatively underexplored. In mice, the MIF pathway controls B‐cell development and the induction of EAE. Here, we found that MIF and MIF receptor CD74 are downregulated, while MIF receptor CXCR4 is upregulated in B cells from early onset MS patients. B cells were identified as the main immune subset in blood expressing MIF. Blocking of MIF and CD74 signaling in B cells triggered CXCR4 expression, and vice versa, with separate effects on their proinflammatory activity, proliferation, and sensitivity to Fas‐mediated apoptosis. This study reveals a new reciprocal negative regulation loop between CD74 and CXCR4 in human B cells. The disturbance of this loop during MS onset provides further insights into how pathogenic B cells survive peripheral tolerance checkpoints to mediate disease activity in MS.

Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in Aicda-/- and Ung-/- Mice.

B cell-depleting therapies have been shown to ameliorate symptoms in multiple sclerosis (MS) patients; however, the mechanism of action remains unclear. Following priming with Ag, B cells undergo secondary diversification of their BCR, including BCR class-switch recombination (CSR) and somatic hypermutation (SHM), with both processes requiring the enzyme activation-induced (cytidine) deaminase. We previously reported that activation-induced (cytidine) deaminase is required for full clinical manifestation of disease in an animal model of MS (experimental autoimmune encephalomyelitis; EAE) provoked by immunization with the extracellular domain of recombinant human myelin oligodendrocyte glycoprotein (hMOG). In this study, we investigated the role of CSR versus SHM in the pathogenesis of EAE. We found that passive transfer of class-switched anti-MOG IgG1 Abs into hMOG-primed Aicda-/- mice is sufficient to fully rescue EAE disease. In addition, we found that the nature of the Ag is an important determinant of EAE severity in Aicda-/- mice because the lack of a diversified BCR does not affect the induction of EAE when immunized with the extracellular domain of rat MOG. To discriminate the effect of either CSR or SHM, we induced EAE in uracil DNA glycosylase-deficient mice (Ung-/-) that exhibit a defect primarily in CSR. We observed that Ung-/- mice exhibit milder clinical disease compared with control mice, concomitant with a reduced amount of anti-MOG IgG1 class-switched Abs that preserved normal affinity. Collectively, these results indicate that CSR plays an important role in governing the incidence and severity of EAE induced with hMOG but not rat MOG.

Obesity results in higher PD-1-mediated T-cell suppression but greater T-cell effector functions following blockade.

65 Background: Obesity is increasingly prevalent and viewed as a critical co-factor in many pathologic conditions due to metabolic, inflammatory and immune perturbations. We performed a multi-species evaluation of the impact of obesity T cell effector functions and markers of immune exhaustion. Methods: We examined the impact of obesity on PD-1 and T cell-mediated responses across different pre-clinical models (tumor, infection, and autoimmune encephalomyelitis [EAE]) and species (mouse, dog, non-human primate, and human). Results: CD4 and CD8 T cells from obese mice, dogs, non-human primates and humans displayed increases in memory T cells and PD-1 expression, as well as impaired proliferative responses compared to lean controls, indicating a greater degree of T cell exhaustion at baseline. Following immunization with myelin oligodendrocyte glycoprotein, obese mice were resistant to induction of EAE, correlating with reduced antigen-specific CD4 T cells in the central nervous system. Administration of anti-PD-1 resulted in restoration of EAE and increased antigen-specific T cell numbers in obese mice. Tumors in obese mice exhibited accelerated growth compared to lean mice, and T cells displayed higher PD-1 expression correlating with RNAseq/molecular signatures of exhaustion compared to tumor-bearing lean mice. PD-1 blockade resulted in marked anti-tumor effects only in obese mice, and not lean. Impaired viral resistance to murine cytomegalovirus (MCMV) resulted was seen in obese mice, associated with increased PD-1/PD-L1 expression, which was reversible by PD-1/PD-L1 blockade. Conclusions: Obesity results in an increase in PD-1/PD-L1 expression and inhibition of T cell responses across species, and blockade not only reverses this inhibition but also leads to markedly augmented T cell effector responses compared to lean counterparts where no effects were observed. These results highlight how the immune system has evolved to control T cell responses using checkpoints contingent on dynamic host conditions and have translational relevance for predicting both efficacy and toxicity in clinical immuno-oncology.

Thrombomodulin‐dependent protein C activation is required for mitochondrial function and myelination in the central nervous system

Background Studies with human samples and in rodents established a function of coagulation proteases in neuro-inflammatory demyelinating diseases (e.g. in multiple sclerosis [MS] and experimental autoimmune encephalitis [EAE]). Surprisingly, approaches to increase activated protein C (aPC) plasma levels as well as antibody-mediated inhibition of PC/aPC ameliorated EAE in mice. Hence, the role of aPC generation in demyelinating diseases and potential mechanisms involved remain controversial. Furthermore, it is not known whether loss of aPC has pathological consequences at baseline (e.g. in the absence of disease). Objective To explore the role of thrombomodulin (TM)-dependent aPC generation at baseline and in immunological and non-immunological demyelinating disease models. Methods Myelination and reactive oxygen species (ROS) generation were evaluated in mice with genetically reduced TM-mediated protein C activation (TMPro/Pro ) and in wild-type (WT) mice under control conditions or following induction of EAE. Non-immunological demyelination was analyzed in the cuprizone-diet model. Results Impaired TM-dependent aPC generation already disturbs myelination and mitochondrial function at baseline. This basal phenotype is linked with increased mitochondrial ROS and aggravates EAE. Reducing mitochondrial ROS (p66Shc deficiency), restoring aPC plasma levels or injecting soluble TM (solulin) ameliorates EAE in TMPro/Pro mice. Soluble TM additionally conveyed protection in WT-EAE mice. Furthermore, soluble TM dampened demyelination in the cuprizone-diet model, demonstrating that its myelin-protective effect is partially independent of an immune-driven process. Conclusion These results uncover a novel physiological function of TM-dependent aPC generation within the CNS. Loss of TM-dependent aPC generation causes a neurological defect in healthy mice and aggravates EAE, which can be therapeutically corrected.

Exposure to inflammatory cytokines selectively limits GM-CSF production by induced T regulatory cells.

Interest in manipulating the immunosuppressive powers of Foxp3-expressing T regulatory cells as an immunotherapy has been tempered by their reported ability to produce proinflammatory cytokines when manipulated in vitro, or in vivo. Understanding processes that can limit this potentially deleterious effect of Treg cells in a therapeutic setting is therefore important. Here, we have studied this using induced (i) Treg cells in which de novo Foxp3 expression is driven by TCR-stimulation in vitro in the presence of TGF-β. We show that iTreg cells can produce significant amounts of three proinflammatory cytokines (IFN-γ, GM-CSF and TNF-α) upon secondary TCR stimulation. GM-CSF is a critical T-cell derived cytokine for the induction of EAE in mice. Despite their apparent capacity to produce GM-CSF, myelin autoantigen-responsive iTreg cells were unable to provoke EAE. Instead, they maintained strong suppressive function in vivo, preventing EAE induction by their CD4+Foxp3− counterparts. We identified that although iTreg cells maintained the ability to produce IFN-γ and TNF-α in vivo, their ability to produce GM-CSF was selectively degraded upon antigen stimulation under inflammatory conditions. Furthermore, we show that IL-6 and IL-27 individually, or IL-2 and TGF-β in combination, can mediate the selective loss of GM-CSF production by iTreg cells.

Conventional housing conditions attenuate the development of experimental autoimmune encephalomyelitis.

BackgroundThe etiology of multiple sclerosis (MS) has remained unclear, but a causative contribution of factors outside the central nervous system (CNS) is conceivable. It was recently suggested that gut bacteria trigger the activation of CNS-reactive T cells and the development of demyelinative disease.MethodsC57BL/6 (B6) mice were kept either under specific pathogen free or conventional housing conditions, immunized with the myelin basic protein (MBP)–proteolipid protein (PLP) fusion protein MP4 and the development of EAE was clinically monitored. The germinal center size of the Peyer’s patches was determined by immunohistochemistry in addition to the level of total IgG secretion which was assessed by ELISPOT. ELISPOT assays were also used to measure MP4-specific T cell and B cell responses in the Peyer’s patches and the spleen. Ear swelling assays were performed to determine the extent of delayed-type hypersensitivity reactions in specific pathogen free and conventionally housed mice.ResultsIn B6 mice that were actively immunized with MP4 and kept under conventional housing conditions clinical disease was significantly attenuated compared to specific pathogen free mice. Conventionally housed mice displayed increased levels of IgG secretion in the Peyer’s patches, while the germinal center formation in the gut and the MP4-specific TH17 response in the spleen were diminished after immunization. Accordingly, these mice displayed an attenuated delayed type hypersensitivity (DTH) reaction in ear swelling assays.ConclusionsThe data corroborate the notion that housing conditions play a substantial role in the induction of murine EAE and suggest that the presence of gut bacteria might be associated with a decreased immune response to antigens of lower affinity. This concept could be of importance for MS and calls for caution when considering the therapeutic approach to treat patients with antibiotics.

Differential IL‐10 production by DCs determines the distinct adjuvant effects of LPS and PTX in EAE induction

Pertussis toxin (PTX) is used as an adjuvant to induce EAE in mice as a model of MS. Although LPS and PTX are similar in their ability to mature dendritic cells (DCs) and in their adjuvant properties, here we demonstrate that LPS does not induce EAE development. We also demonstrate that DCs treated with PTX (PTX-DCs) are able to induce EAE, whereas DCs treated with LPS (LPS-DCs) fail to induce EAE. We determine that a key difference between LPS-DCs and PTX-DCs is that LPS-DCs produce larger amounts of IL-10. IL-10(-/-) -DCs treated with LPS promote stronger IFN-γ and IL-17 production and T-cell proliferation than WT DCs treated with LPS in a coculture system. Finally, we demonstrate that EAE can be successfully induced when IL-10(-/-) -DCs treated with LPS are used as an adjuvant, whereas the use of PTX-DCs overexpressing IL-10 as an adjuvant markedly controls EAE development. These results indicate that the inability of LPS to induce EAE is based on the induction of high levels of IL-10 in the targeted DCs, providing insight into the mechanisms responsible for the induction of EAE.

A novel aza-anthrapyrazole modulates the development of CD4+ effector T cells (P5207)

Abstract BBR3378 is a novel aza-anthrapyrazole that antagonizes autoimmunity and the progression of MOG-induced EAE without the complications noted with anthracycline-based (mitoxantrone) therapy in EAE and human multiple sclerosis. Mitoxantrone induces cell death in rapidly proliferating cells and significant lymphopenia in vivo; however, while most splenic CD4+ cells take up BBR3378 in vivo, only a few are deleted. Moreover, increased dosages of BBR3378 were associated with improved therapeutic impact without concomitant increases in lymphopenia. To determine if BBR3378-mediates regulation of EAE via lymphocyte deletion, splenic T cells were stimulated with anti-CD3 in the presence of low-dose BBR3378 or mitoxantrone. The CD3 blasts showed increased expression of CD25 and CD69, but the enhanced CD4 expression (CD4hi) typically associated with Th progressison to “effector” status was blocked on BBR3378 and mitoxantrone-treated cells, leading to enrichment of slowly proliferating CD4lo-CD69hi T cells. Cytokine production was confined to CD4hi-CD69hi blasts; however, in contrast to mitoxantrone, IL-17 and IFN-gamma production were significantly reduced in the BBR3378-treated cells in the absence of Th cell death. These findings suggest that BBR3378 can regulate specific genes in viable Th cells while impeding the progression of naïve Th cells to full effector status. The Th subsets associated with the induction of EAE were particularly sensitive to BBR3378-mediated suppression.

IL-17A secretion by CD8+ T cells supports Th17-mediated autoimmune encephalomyelitis

IL-17-producing CD8+ T (Tc17) cells are detectible in multiple sclerosis (MS) lesions; however, their contribution to the disease is unknown. To identify functions of Tc17 cells, we induced EAE, a murine model of MS, in mice lacking IFN regulatory factor 4 (IRF4). IRF4-deficient mice failed to generate Tc17 and Th17 cells and were resistant to EAE. After adoptive transfer of WT CD8+ T cells and subsequent immunization for EAE induction in these mice, the CD8+ T cells developed a Tc17 phenotype in the periphery but could not infiltrate the CNS. Similarly, transfer of small numbers of WT CD4+ T cells alone did not evoke EAE, but when transferred together with CD8+ T cells, IL-17-producing CD4+ (Th17) T cells accumulated in the CNS and mice developed severe disease. Th17 accumulation and development of EAE required IL-17A production by CD8+ T cells, suggesting that Tc17 cells are required to promote CD4+ T cell-mediated induction of EAE. Accordingly, patients with early-stage MS harbored a greater number of Tc17 cells in the cerebrospinal fluid than in peripheral blood. Our results reveal that Tc17 cells contribute to the initiation of CNS autoimmunity in mice and humans by supporting Th17 cell pathogenicity.

Transgenic inhibition of astroglial NF-κB protects from optic nerve damage and retinal ganglion cell loss in experimental optic neuritis

BackgroundOptic neuritis is an acute, demyelinating neuropathy of the optic nerve often representing the first appreciable symptom of multiple sclerosis. Wallerian degeneration of irreversibly damaged optic nerve axons leads to death of retinal ganglion cells, which is the cause of permanent visual impairment. Although the specific mechanisms responsible for triggering these events are unknown, it has been suggested that a key pathological factor is the activation of immune-inflammatory processes secondary to leukocyte infiltration. However, to date, there is no conclusive evidence to support such a causal role for infiltrating peripheral immune cells in the etiopathology of optic neuritis.MethodsTo dissect the contribution of the peripheral immune-inflammatory response versus the CNS-specific inflammatory response in the development of optic neuritis, we analyzed optic nerve and retinal ganglion cells pathology in wild-type and GFAP-IκBα-dn transgenic mice, where NF-κB is selectively inactivated in astrocytes, following induction of EAE.ResultsWe found that, in wild-type mice, axonal demyelination in the optic nerve occurred as early as 8 days post induction of EAE, prior to the earliest signs of leukocyte infiltration (20 days post induction). On the contrary, GFAP-IκBα-dn mice were significantly protected and showed a nearly complete prevention of axonal demyelination, as well as a drastic attenuation in retinal ganglion cell death. This correlated with a decrease in the expression of pro-inflammatory cytokines, chemokines, adhesion molecules, as well as a prevention of NAD(P)H oxidase subunit upregulation.ConclusionsOur results provide evidence that astrocytes, not infiltrating immune cells, play a key role in the development of optic neuritis and that astrocyte-mediated neurotoxicity is dependent on activation of a transcriptional program regulated by NF-κB. Hence, interventions targeting the NF-κB transcription factor in astroglia may be of therapeutic value in the treatment of optic neuritis associated with multiple sclerosis.

Interferon regulatory factor-7 modulates experimental autoimmune encephalomyelitis in mice

BackgroundMultiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) with unknown etiology. Interferon-β (IFN-β), a member of the type I IFN family, is used as a therapeutic for MS and the IFN signaling pathway is implicated in MS susceptibility. Interferon regulatory factor 7 (IRF7) is critical for the induction and positive feedback regulation of type I IFN. To establish whether and how endogenous type I IFN signaling contributes to disease modulation and to better understand the underlying mechanism, we examined the role of IRF7 in the development of MS-like disease in mice.MethodsThe role of IRF7 in development of EAE was studied by immunizing IRF7-KO and C57BL/6 (WT) mice with myelin oligodendrocyte glycoprotein using a standard protocol for the induction of EAE. We measured leukocyte infiltration and localization in the CNS using flow cytometric analysis and immunohistochemical procedures. We determined levels of CD3 and selected chemokine and cytokine gene expression by quantitative real-time PCR.ResultsIRF7 gene expression increased in the CNS as disease progressed. IRF7 message was localized to microglia and infiltrating leukocytes. Furthermore, IRF7-deficient mice developed more severe disease. Flow cytometric analysis showed that the extent of leukocyte infiltration into the CNS was higher in IRF7-deficient mice with significantly higher number of infiltrating macrophages and T cells, and the distribution of infiltrates within the spinal cord was altered. Analysis of cytokine and chemokine gene expression by quantitative real-time PCR showed significantly greater increases in CCL2, CXCL10, IL-1β and IL17 gene expression in IRF7-deficient mice compared with WT mice.ConclusionTogether, our findings suggest that IRF7 signaling is critical for regulation of inflammatory responses in the CNS.

Polyclonal Treg cells modulate T effector cell trafficking

In this study, we have analyzed the in vivo dynamics of the interaction between polyclonal Foxp3(+) Treg cells, effector T (Teff) cells, and DCs in order to further our understanding of the mechanisms of Treg cell-mediated suppression. Cotransfer of polyclonal activated Treg cells into healthy mice attenuated the induction of EAE. Suppression of disease strongly correlated with a reduced number of Teff cells in the spinal cord, but not with Treg cell-mediated inhibition of Th1/Th17 differentiation. Cotransfer of Treg cells with TCR-Tg Teff cells followed by immunization by multiple routes resulted in an enhanced number of Teff cells in the lymph nodes draining the site of immunization without an inhibition of Teff-cell differentiation. Fewer Teff cells could be detected in the blood in the presence of Treg cells and fewer T cells could access a site of antigen exposure in a modified delayed-type hypersensitivity assay. Teff cells recovered from LNs in the presence of Treg cells expressed decreased levels of CXCR4, syndecan, and the sphingosine phosphate receptor, S1P1 (sphingosine 1-phosphate receptor 1). Thus, polyclonal Treg cells influence Teff-cell responses by targeting trafficking pathways, thus allowing immunity to develop in lymphoid organs, but limiting the number of potentially auto-aggressive cells that are allowed to enter the tissues.

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Cytokine-neuroantigen fusion proteins represent a novel therapeutic approach for EAE (48.10)

Abstract Cytokine-neuroantigen (NAg) fusion proteins have promise as a new class of antigen-specific therapies for treatment of CNS autoimmune disease. The primary objective of this study was to assess therapeutic efficacy of selected cytokine-neuroantigen fusion proteins in the Lewis rat model of EAE. The neuroantigen domain of each fusion protein was comprised of the major encephalitogenic determinant of myelin basic protein. Three cytokine-NAg fusion proteins were potent inhibitors of EAE. These fusion proteins incorporated the IL-2 cytokine as the N-terminal domain (the IL2NAg fusion protein), the secreted IL-16 cytokine as the C-terminal domain (the NAgIL16 fusion protein), or the IFN-beta cytokine as the N-terminal domain (IFNbeta-NAg fusion protein). Compared to NAg alone, these fusion proteins strongly attenuated the subsequent active induction of EAE. These fusion proteins also inhibited EAE when administered after encephalitogenic challenge during onset of EAE. In conclusion, cytokine-NAg fusion proteins that incorporate IL-2, IL-16, or IFN-beta cytokine domains may comprise an effective tolerogenic therapy for CNS autoimmune disease.

Signaling by the serine/threonine kinase Drak2 in T cells (35.23)

Abstract A role for Drak2 in negative regulation of T cell activation has been well established, yet the manner in which Drak2 mediates downstream events to limit the spurious activation of T cells remains to be elucidated. While T cells from mice deficient in Drak2 exhibit a decreased activation threshold and hyper-respond to sub-optimal stimuli, the proliferative capacity of these cells is diminished as result of a deficiency in survival. A resistance to the induction of EAE in Drak2-deficient mice underscores a dependence on negative regulatory molecules to enforce the requirement of co-stimulation to engage an effective immune response. Biochemical data tracking Drak2 activation has revealed that protein kinase D (PKD) is required for Drak2 signaling. PKD has been shown to respond to the release of calcium from intracellular stores as well as to oxidative stress, two events coupled to T cell activation through the processes of IP3R-mediated ER-calcium depletion and increased mitochondrial respiration. The signaling requirements of Drak2 similarly include both store-operated calcium entry and the production of reactive oxygen species. The data presented indicate that Drak2 and PKD constitute a signaling axis that defines a novel role for PKD in T cell activation and further sheds light on how Drak2 orchestrates decreased responsiveness as a negative regulatory molecule.

Site‐specific targeting of autoimmunity in mice induced by the localized production of IL‐6

IL‐6 is required for the induction of experimental autoimmunity. The role of IL‐6 in EAE was examined in transgenic mice (GF‐IL6) with cerebellum specific production of IL‐6. MOG‐immunized (Mi‐) GF‐IL6 mice developed severe ataxia which was in contrast to Mi‐wild type (WT) mice that developed classical EAE with hind limb paralysis. Immune pathology and demyelination were markedly decreased in the spinal cord but significantly increased in the cerebellum of Mi‐GF‐IL6 mice. Tissue damage in the cerebellum at peak disease in the Mi‐GF‐IL6 mice was accompanied by increased total numbers of infiltrating leukocytes and increased proportions of both neutrophils and B‐cells. During peak disease, the expression of various cytokines was found at comparable levels in the cerebellum between Mi‐GF‐IL6 and Mi‐WT, whereas significantly higher levels were found in Mi‐WT spinal cord. In conclusion, site‐specific production of IL‐6 targets and modifies the autoreactive inflammatory response and creates a leukocyte “sink” that retards trafficking to normally dominant antigenic sites. The mechanisms underlying this response to IL‐6 likely include the induction of specific chemokines as well as increased activation and loss of integrity of the BBB in the cerebellum of the GF‐IL6 mice observed prior to the induction of EAE. Support:NSW Spinal Cord Injury and Other Neurological Conditions Project Grant.

A role for mast cells in EAE: W/Wv, Wsh, and CD34−/− mice are fully susceptible to disease induction using standard protocols (B92)

Abstract EAE is a murine model of multiple sclerosis involving destruction of myelin by the immune system. Mast cells, known for a role in asthma and allergy, are reportedly required for EAE induction in mice. Our lab has reported that mature mast cells express the surface marker CD34. To further understand the role of mast cells in EAE, we utilized CD34−/− mice which have functional mast cells in the tissues, but are defective in their mobilization. We asked whether mast cells seeded in development were sufficient for EAE induction. Previous studies of mast cells in EAE used W/Wv mice, where reconstitution with WT-mast cells restored susceptibility to EAE. It was possible that CD34−/− mice were protected from EAE due to the decreased peripheral mobility of mast cells, or, seeding of CNS- or vascular-localized mast cells during development could be sufficient for their function. Here we compare clinical disease in WT, CD34−/−, W/Wv, and Wsh mice. We find CD34−/− mice are susceptible to induction of EAE, consistent with suggestions that mast cells act remotely in this model. Surprisingly, however, we also find both W/Wv and Wsh mice susceptible to disease in our hands. This result was confirmed by other laboratories. Interestingly, analysis of infiltrating leukocytes in the CD34−/− CNS shows increased mast cells vs. WT. We are now investigating whether inflammatory factors have bypassed the requirement for cKit on mast cells in EAE.