Exacerbation of experimental autoimmune encephalomyelitis in P2X7R-/- mice: evidence for loss of apoptotic activity in lymphocytes

The purinergic receptor P2X7R is a nucleotide-gated ion channel that has been proposed to function as a major regulator of inflammation. In this study we examined the role of this receptor in regulating inflammation in the CNS by determining the effects of the loss of this receptor (P2X7R-/-) on experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. We show here that P2X7R-/- mice developed more severe clinical and pathological expression of EAE than wild type (WT) controls and that spleen and lymph node cells from P2X7R-/- mice proliferated more vigorously to Ag in vitro. Bone marrow (BM) radiation chimeras revealed that enhanced susceptibility to EAE was detected in chimeric mice of WT host engrafted with P2X7R-/- BM cells, indicating that the genotype of the BM cells regulated disease susceptibility. Coculture of P2X7R-/- macrophages with WT lymphocytes and vice versa showed that enhanced proliferative activity resided within the P2X7R-/- lymphocyte population and correlated with reduced levels of IFN-gamma and NO and apoptosis of lymphocytes. mRNA and protein for IFN-gamma were also significantly reduced in the CNS of P2X7R-/- mice with EAE. FACS analysis of cells isolated from the CNS showed significantly fewer annexin V/propidium iodide-positive lymphocytes in the CNS of P2X7R-/- mice early in the disease, and TUNEL staining of inflamed CNS tissues supported this result. From these data we conclude that enhanced susceptibility of P2X7R-/- mice to EAE reflects a loss of apoptotic activity in lymphocytes, supporting an important role for this receptor in lymphocyte homeostasis.     

Corticotropin-releasing hormone contributes to the peripheral inflammatory response in experimental autoimmune encephalomyelitis

Peripheral corticotropin-releasing hormone (CRH) is thought to have proinflammatory effects. We used the model of experimental autoimmune encephalomyelitis (EAE) to study the role of CRH in an immune-mediated disease. We showed that CRH-deficient mice are resistant to EAE, with a decrease in clinical score as well as decreased cellular infiltration in the CNS. Furthermore, Ag-specific responses of primed T cells as well as anti-CD3/anti-CD28 TCR costimulation were decreased in crh(-/-) mice with decreased production of Th1 cytokines and increased production of Th2 cytokines. Wild-type mice treated in vivo with a CRH antagonist showed a decrease in IFN-gamma production by primed T cells in vitro. This effect of CRH is independent of its ability to increase corticosterone production, because adrenalectomized wild-type mice had similar disease course and severity as control mice. We found that IkappaBalpha phosphorylation induced by TCR cross-linking was decreased in crh(-/-) T cells. We conclude that peripheral CRH exerts a proinflammatory effect in EAE with a selective increase in Th1-type responses. These findings have implications for the treatment of Th1-mediated diseases such as multiple sclerosis.     

Epithelial V-Like Antigen Mediates Efficacy of Anti-Alpha4 Integrin Treatment in a Mouse Model of Multiple Sclerosis

Natalizumab inhibits the transmigration of activated T lymphocytes into the brain and is highly efficacious in multiple sclerosis (MS). However, from a pharmacogenomic perspective, its efficacy and safety in specific patients remain unclear. Here our goal was to analyze the effects of epithelial V-like antigen (EVA) on anti-alpha₄ integrin (VLA4) efficacy in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). EVA has been previously characterized in human CD4 T lymphocytes, mouse thymic development, and choroid plexus epithelial cells. Further analysis here demonstrated expression in B lymphocytes and an increase in EVA⁺ lymphocytes following immunization. Following active induction of EAE using the MOG^35–55 active immunization model, EVA deficient mice developed more severe EAE and white matter tissue injury as compared to wild type controls. This severe EAE phenotype did not respond to anti-VLA4 treatment. In both the control antibody and anti-VLA4 conditions, these mice demonstrated persistent CNS invasion of mature B lymphocyte (CD19⁺, CD21⁺, sIgG⁺), increased serum autoantibody levels, and extensive complement and IgG deposition within lesions containing CD5⁺IgG⁺ cells. Wild type mice treated with control antibody also demonstrated the presence of CD19⁺, CD21⁺, sIgG⁺ cells within the CNS during peak EAE disease severity and detectable serum autoantibody. In contrast, wild type mice treated with anti-VLA4 demonstrated reduced serum autoantibody levels as compared to wild type controls and EVA-knockout mice. As expected, anti-VLA4 treatment in wild type mice reduced the total numbers of all CNS mononuclear cells and markedly decreased CD4 T lymphocyte invasion. Treatment also reduced the frequency of CD19⁺, CD21⁺, sIgG⁺ cells in the CNS. These results suggest that anti-VLA4 treatment may reduce B lymphocyte associated autoimmunity in some individuals and that EVA expression is necessary for an optimal therapeutic response. We postulate that these findings could optimize the selection of treatment responders.     

Intrathecal delivery of IFN-gamma protects C57BL/6 mice from chronic-progressive experimental autoimmune encephalomyelitis by increasing apoptosis of central nervous system-infiltrating lymphocytes

The exclusive detrimental role of proinflammatory cytokines in demyelinating diseases of the CNS, such as multiple sclerosis, is controversial. Here we show that the intrathecal delivery of an HSV-1-derived vector engineered with the mouse IFN-gamma gene leads to persistent (up to 4 wk) CNS production of IFN-gamma and inhibits the course of a chronic-progressive form of experimental autoimmune encephalomyelitis (EAE) induced in C57BL/6 mice by myelin oligodendrocyte glycoprotein (MOG)(35-55). Mice treated with the IFN-gamma-containing vector before EAE onset showed an earlier onset but a milder course of the disease compared with control mice treated with the empty vector. In addition, 83% of IFN-gamma-treated mice completely recovered within 25 days post immunization, whereas control mice did not recover up to 60 days post immunization. Mice treated with the IFN-gamma-containing vector within 1 wk after EAE onset partially recovered from the disease within 25 days after vector injection, whereas control mice worsened. Recovery from EAE in mice treated with IFN-gamma was associated with a significant increase of CNS-infiltrating lymphocytes undergoing apoptosis. During the recovery phase, the mRNA level of TNFR1 was also significantly increased in CNS-infiltrating cells from IFN-gamma-treated mice compared with controls. Our results further challenge the exclusive detrimental role of IFN-gamma in the CNS during EAE/multiple sclerosis, and indicate that CNS-confined inflammation may induce protective immunological countermechanisms leading to a faster clearance of encephalitogenic T cells by apoptosis, thus restoring the immune privilege of the CNS.     

Severe disease, unaltered leukocyte migration, and reduced IFN-gamma production in CXCR3-/- mice with experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) Th1 T cell-mediated disease of the CNS, used to study certain aspects of multiple sclerosis. CXCR3, the receptor for CXCL10, CXCL9, and CXCL11, is preferentially expressed on activated Th1 T cells and has been proposed to govern the migration of lymphocytes into the inflamed CNS during multiple sclerosis and EAE. Unexpectedly, CXCL10-deficient mice were susceptible to EAE, leaving uncertain what the role of CXCR3 and its ligands might play in this disease model. In this study, we report that CXCR3(-/-) mice exhibit exaggerated severity of EAE compared with wild-type (CXCR3(+/+)) littermate mice. Surprisingly, there were neither quantitative nor qualitative differences in CNS-infiltrating leukocytes between CXCR3(+/+) and CXCR3(-/-) mice with EAE. Despite these equivalent inflammatory infiltrates, CNS tissues from CXCR3(-/-) mice with EAE showed worsened blood-brain barrier disruption and more von Willebrand factor-immunoreactive vessels within inflamed spinal cords, as compared with CXCR3(+/+) mice. Spinal cords of CXCR3(-/-) mice with EAE demonstrated decreased levels of IFN-gamma, associated with reduced inducible NO synthase immunoreactivity, and lymph node T cells from CXCR3(-/-) mice primed with MOG(35-55) secreted less IFN-gamma in Ag-driven recall responses than cells from CXCR3(+/+) animals. CXCR3(-/-) lymph node T cells also showed enhanced Ag-driven proliferation, which was reduced by addition of IFN-gamma. Taken with prior findings, our data show that CXCL10 is the most relevant ligand for CXCR3 in EAE. CXCR3 does not govern leukocyte trafficking in EAE but modulates T cell IFN-gamma production and downstream events that affect disease severity.     

Analysis of protein induction in the CNS of SJL mice with experimental allergic encephalomyelitis by proteomic screening and immunohistochemistry.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterized by demyelination and inflammatory infiltrates in the CNS, and it is an animal model of multiple sclerosis. Piperonyl butoxide (PBO) suppresses disease in EAE mice, and it exhibits a dual effect on cytochrome P450s that manifests in a transient inhibitory phase followed by induction. In order to identify the expression of proteins associated with EAE, a proteomic screening was performed on hindbrain microsomes from control + vehicle, control + PBO, EAE + vehicle, and EAE + PBO female mice. Glucose regulated protein 94 (Grp94) and coagulation factor VIII were among the proteins identified in EAE + vehicle and EAE + PBO mice. Immunohistochemical staining of Grp94 was present in some neurons and oligodendrocytes in hindbrain sections from control animals, and in some cells within inflammatory infiltrates in EAE animals. Since Grp94 (also known as Gp96) can partake in antigen presentation and induction of proinflammatory cytokine expression, its presence in these cells suggests that it may play a role in the pathogenesis of EAE. Coagulation factor VIII is carried and protected by von Willebrand factor. Immunohistochemical staining of von Willebrand factor revealed its presence in some vessels within hindbrain sections from control animals. In EAE animals, the number of labeled vessels was significantly increased, and extracellular granular deposits were observed around labeled vessels indicating that the breakdown of the blood-brain barrier that occurs in EAE permitted its extravasation into the CNS. Additional proteins were identified in the different groups of mice by proteomic screening, but confirmation of their expression profile awaits investigations by independent measures.     

IFN-inducible protein 10/CXC chemokine ligand 10-independent induction of experimental autoimmune encephalomyelitis

In multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), autoaggressive T cells traffic into the CNS and induce disease. Infiltration of these pathogenic T cells into the CNS has been correlated with the expression of the chemokine IFN-inducible protein (IP)10/CXC chemokine ligand (CXCL)10, a chemoattractant for activated T cells, and its receptor CXCR3, in the CNS of both MS patients and mice with EAE. In the present study, we report that targeted deletion of IP-10 did not diminish the expression, severity, or histopathology of EAE induced by active immunization with 100 micro g of myelin oligodendrocyte glycoprotein peptide (MOG)p35-55. However, we found that IP-10-deficient mice had a lower threshold for expression of disease compared with wild-type littermates. EAE induced by immunization with 5 micro g of MOGp35-55 resulted in more severe disease characterized by a greater number of CNS lesions and infiltrating mononuclear cells in IP-10-deficient mice compared with wild-type controls. IP-10-deficient mice immunized with MOGp35-55 demonstrated increased levels of IFN-inducible T cell alpha-chemokine/CXCL11 mRNA in the CNS and decreased levels of monokine induced by IFN-gamma/CXCL9 mRNA in draining lymph nodes, suggesting differential compensation for loss of IP-10 in lymphoid vs parenchymal tissue compartments. EAE in IP-10-deficient mice induced by low-dose immunization was associated with enhanced Ag-specific Th1 responses in the draining lymph node, which corresponded with diminished lymph node TGF-beta1 expression. Our data demonstrated that IP-10 was not required for the trafficking of pathogenic T cells into the CNS in EAE but played an unexpected role in determining the threshold of disease susceptibility in the periphery.     

Reduced chemokine and chemokine receptor expression in spinal cords of TCR BV8S2 transgenic mice protected against experimental autoimmune encephalomyelitis with BV8S2 protein

The perivascular transmigration and accumulation of macrophages and T lymphocytes in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) may be partly regulated by low m.w. chemotactic cytokines. Using the RNase protection assay and ELISA, we quantified expression of chemokines and chemokine receptors in the spinal cord (SC), brain, and lymph nodes of BV8S2 transgenic mice that developed or were protected from EAE by vaccination with BV8S2 protein. In paralyzed control mice, the SC had increased cellular infiltration and strong expression of the chemokines RANTES, IFN-inducible 10-kDa protein, and monocyte chemoattractant protein-1 and the cognate chemokine receptors CCR1, CCR2, and CCR5, with lower expression of macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, and MIP-2; whereas brain had less infiltration and a lower expression of a different pattern of chemokines and receptors. In TCR-protected mice, there was a decrease in the number of inflammatory cells in both SC and brain. In SC, the reduced cellular infiltrate afforded by TCR vaccination was commensurate with profoundly reduced expression of chemokines and their cognate chemokine receptors. In brain, however, TCR vaccination did not produce significant changes in chemokine expression but resulted in an increased expression of CCR3 and CCR4 usually associated with Th2 cells. In contrast to CNS, lymph nodes of protected mice had a significant increase in expression of MIP-2 and MIP-1beta but no change in expression of chemokine receptors. These results demonstrate that TCR vaccination results in selective reduction of inflammatory chemokines and chemokine receptors in SC, the target organ most affected during EAE.     

Sodium benzoate, a food additive and a metabolite of cinnamon, modifies T cells at multiple steps and inhibits adoptive transfer of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is the animal model for multiple sclerosis. This study explores a novel use of sodium benzoate (NaB), a commonly used food additive and a Food and Drug Administration-approved nontoxic drug for urea cycle disorders, in treating the disease process of relapsing-remitting EAE in female SJL/J mice. NaB, administered through drinking water at physiologically tolerable doses, ameliorated clinical symptoms and disease progression of EAE in recipient mice and suppressed the generation of encephalitogenic T cells in donor mice. Histological studies reveal that NaB effectively inhibited infiltration of mononuclear cells and demyelination in the spinal cord of EAE mice. Consequently, NaB also suppressed the expression of proinflammatory molecules and normalized myelin gene expression in the CNS of EAE mice. Furthermore, we observed that NaB switched the differentiation of myelin basic protein-primed T cells from Th1 to Th2 mode, enriched regulatory T cell population, and down-regulated the expression of various contact molecules in T cells. Taken together, our results suggest that NaB modifies encephalitogenic T cells at multiple steps and that NaB may have therapeutic importance in multiple sclerosis.     

Oral feeding with ethinyl estradiol suppresses and treats experimental autoimmune encephalomyelitis in SJL mice and inhibits the recruitment of inflammatory cells into the central nervous system

There is much interest in the possible ameliorating effects of estrogen on various autoimmune diseases. We previously established the protective effects of 17 beta-estradiol (E2) on experimental autoimmune encephalomyelitis (EAE). In the current study we investigated the effectiveness of oral treatment with ethinyl estradiol (EE) on EAE and the mechanisms involved. Ethinyl estradiol is a semisynthetic estrogen compound found in birth control pills, and its chemical structure allows this compound to retain activity when given orally. We found that oral EE, like E2, drastically suppressed EAE induced by proteolipid protein 139-151 peptide when given at initiation of EAE. However, unlike E2, EE reduced clinical severity when given after the onset of clinical signs. Treatment with EE significantly decreased the secretion of proinflammatory cytokines (IFN-gamma, TNF-alpha, and IL-6) by activated T cells as well as the expression of a key matrix metalloproteinase, disease-mediating chemokines/receptors, and IgG2a levels, but increased the expression of TGF-beta 3 in the CNS. The absence of infiltrating lymphocytes together with the suppression of cytokines, matrix metalloproteinase, and chemokines/receptors suggests that EE, like E2, protects mice from EAE by inhibiting the recruitment of T cells and macrophages into the CNS. These results suggest that oral ethinyl estradiol might be a successful candidate as therapy for multiple sclerosis.     

Efficient recruitment of lymphocytes in inflamed brain venules requires expression of cutaneous lymphocyte antigen and fucosyltransferase-VII

Lymphocyte migration into the brain represents a critical event in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). However, the mechanisms controlling the recruitment of lymphocytes to the CNS via inflamed brain venules are poorly understood, and therapeutic approaches to inhibit this process are consequently few. In this study, we demonstrate for the first time that human and murine Th1 lymphocytes preferentially adhere to murine inflamed brain venules in an experimental model that mimics early inflammation during EAE. A virtually complete inhibition of rolling and arrest of Th1 cells in inflamed brain venules was observed with a blocking anti-P-selectin glycoprotein ligand 1 Ab and anti-E- and P-selectin Abs. Th1 lymphocytes produced from fucosyltransferase (FucT)-IV(-/-) mice efficiently tethered and rolled, whereas in contrast, primary adhesion of Th1 lymphocytes obtained from FucT-VII(-/-) or Fuc-VII(-/-)FucT-IV(-/-) mice was drastically reduced, indicating that FucT-VII is critical for the recruitment of Th1 cells in inflamed brain microcirculation. Importantly, we show that Abs directed against cutaneous lymphocyte Ag (CLA), a FucT-VII-dependent carbohydrate modification of P-selectin glycoprotein ligand 1, blocked rolling of Th1 cells. By exploiting a system that allowed us to obtain Th1 and Th2 cells with skin- vs gut-homing (CLA(+) vs integrin beta(7)(+)) phenotypes, we observed that induced expression of CLA on Th cells determined a striking increase of rolling efficiency in inflamed brain venules. These observations allow us to conclude that efficient recruitment of activated lymphocytes to the brain in the contexts mimicking EAE is controlled by FucT-VII and its cognate cell surface Ag CLA.     

Host T cells are the main producers of IL-17 within the central nervous system during initiation of experimental autoimmune encephalomyelitis induced by adoptive transfer of Th1 cell lines

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, has long been thought to be mediated by Th1 CD4(+) T cells. Using adoptive transfer techniques, transfer of CNS specific Th1 T cells was sufficient to induce EAE in naive mice. However, recent studies found a vital role for IL-17 in induction of EAE. These studies suggested that a fraction of IL-17-producing T cells that contaminate Th1 polarized cell lines are largely responsible for initiation of EAE. In this study, we tracked the appearance and cytokine production capacity of adoptively transferred cells within the CNS of mice throughout EAE disease. IL-17-producing, adoptively transferred cells were not enriched over the low percentages present in vitro. Thus, there was no selective recruitment and/or preferential proliferation of adoptively transferred IL-17-producing cells during the induction of EAE. Instead a large number of CNS infiltrating host T cells in mice with EAE were capable of producing IL-17 following ex vivo stimulation. The IL-17-producing T cells contained both alphabeta and gammadelta TCR(+) T cells with a CD4(+)CD8(-) or CD4(-)CD8(-) phenotype. These cells concentrated within the CNS within 3 days of adoptive transfer, and appeared to play a role in EAE induction as adoptive transfer of Th1 lines derived from wild-type mice into IL-17-deficient mice induced reduced EAE clinical outcomes. This study demonstrates that an encephalitogenic Th1 cell line induces recruitment of host IL-17-producing T cells to the CNS during the initiation of EAE and that these cells contribute to the incidence and severity of disease.     

Adenosine A2A receptor activation reduces infarct size in the isolated, perfused mouse heart by inhibiting resident cardiac mast cell degranulation

Mast cells are found in the heart and contribute to reperfusion injury following myocardial ischemia. Since the activation of A2A adenosine receptors (A2AARs) inhibits reperfusion injury, we hypothesized that ATL146e (a selective A2AAR agonist) might protect hearts in part by reducing cardiac mast cell degranulation. Hearts were isolated from five groups of congenic mice: A2AAR+/+ mice, A2AAR(-/-) mice, mast cell-deficient (Kit(W-sh/W-sh)) mice, and chimeric mice prepared by transplanting bone marrow from A2AAR(-/-) or A2AAR+/+ mice to radiation-ablated A2AAR+/+ mice. Six weeks after bone marrow transplantation, cardiac mast cells were repopulated with >90% donor cells. In isolated, perfused hearts subjected to ischemia-reperfusion injury, ATL146e or CGS-21680 (100 nmol/l) decreased infarct size (IS; percent area at risk) from 38 +/- 2% to 24 +/- 2% and 22 +/- 2% in ATL146e- and CGS-21680-treated hearts, respectively (P < 0.05) and significantly reduced mast cell degranulation, measured as tryptase release into reperfusion buffer. These changes were absent in A2AAR(-/-) hearts and in hearts from chimeric mice with A2AAR(-/-) bone marrow. Vehicle-treated Kit(W-sh/W-sh) mice had lower IS (11 +/- 3%) than WT mice, and ATL146e had no significant protective effect (16 +/- 3%). These data suggest that in ex vivo, buffer-perfused hearts, mast cell degranulation contributes to ischemia-reperfusion injury. In addition, our data suggest that A2AAR activation is cardioprotective in the isolated heart, at least in part by attenuating resident mast cell degranulation.     

A Novel CMKLR1 Small Molecule Antagonist Suppresses CNS Autoimmune Inflammatory Disease

Therapies that target leukocyte trafficking pathways can reduce disease activity and improve clinical outcomes in multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) is a widely studied animal model that shares many clinical and histological features with MS. Chemokine-like receptor-¹ (CMKLR1) is a chemoattractant receptor that is expressed by key effector cells in EAE and MS, including macrophages, subsets of dendritic cells, natural killer cells and microglia. We previously showed that CMKLR1-deficient (CMKLR1 KO) mice develop less severe clinical and histological EAE than wild-type mice. In this study, we sought to identify CMKLR1 inhibitors that would pharmaceutically recapitulate the CMKLR1 KO phenotype in EAE. We identified 2-(α-naphthoyl) ethyltrimethylammonium iodide (α-NETA) as a CMKLR1 small molecule antagonist that inhibits chemerin-stimulated β-arrestin2 association with CMKLR1, as well as chemerin-triggered CMKLR1⁺ cell migration. α-NETA significantly delayed the onset of EAE induced in C57BL/6 mice by both active immunization with myelin oligodendrocyte glycoprotein peptide 35-55 and by adoptive transfer of encephalitogenic T cells. In addition, α-NETA treatment significantly reduced mononuclear cell infiltrates within the CNS. This study provides additional proof-of-concept data that targeting CMKLR1:chemerin interactions may be beneficial in preventing or treating MS.     

De novo central nervous system processing of myelin antigen is required for the initiation of experimental autoimmune encephalomyelitis

We demonstrate the absolute requirement for a functioning class II-restricted Ag processing pathway in the CNS for the initiation of experimental autoimmune encephalomyelitis (EAE). C57BL/6 (B6) mice deficient for the class II transactivator, which have defects in MHC class II, invariant chain (Ii), and H-2M (DM) expression, are resistant to initiation of myelin oligodendrocyte protein (MOG) peptide, MOG(35-55)-specific EAE by both priming and adoptive transfer of encephalitogenic T cells. However, class II transactivator-deficient mice can prime a suboptimal myelin-specific CD4(+) Th1 response. Further, B6 mice individually deficient for Ii and DM are also resistant to initiation of both active and adoptive EAE. Although both Ii-deficient and DM-deficient APCs can present MOG peptide to CD4(+) T cells, neither is capable of processing and presenting the encephalitogenic peptide of intact MOG protein. This phenotype is not Ag-specific, as DM- and Ii-deficient mice are also resistant to initiation of EAE by proteolipid protein peptide PLP(178-191). Remarkably, DM-deficient mice can prime a potent peripheral Th1 response to MOG(35-55), comparable to the response seen in wild-type mice, yet maintain resistance to EAE initiation. Most striking is the demonstration that T cells from MOG(35-55)-primed DM knockout mice can adoptively transfer EAE to wild-type, but not DM-deficient, mice. Together, these data demonstrate that the inability to process antigenic peptide from intact myelin protein results in resistance to EAE and that de novo processing and presentation of myelin Ags in the CNS is absolutely required for the initiation of autoimmune demyelinating disease.     

Gamma delta T cells regulate the extent and duration of inflammation in the central nervous system by a Fas ligand-dependent mechanism

Gamma delta T cells have been shown to regulate immune responses associated with inflammation, but the mechanism of this regulation is largely unknown. Using the experimental autoimmune encephalomyelitis (EAE) model of the human CNS autoimmune disease multiple sclerosis, we demonstrate that gamma delta T cells are important regulators of CNS inflammation. This was shown using gamma delta T cell-deficient mice that were unable to recover from EAE. The chronic disease was accompanied by a prolonged presence of both macrophages and lymphocytes in the CNS. This extended inflammatory response was due to alterations in both cell proliferation and death. In mice lacking gamma delta T cells, proliferation of encephalitogenic T cells was 3-fold higher, and caspase activity, indicating apoptosis, was 2-fold lower compared with those in control mice recovering from EAE. gamma delta T cell-deficient mice reconstituted with wild-type gamma delta T cells recovered from EAE and resolved inflammation in the CNS, whereas mice reconstituted with Fas ligand-dysfunctional gamma delta T cells did not. Thus, gamma delta T cells regulate both inflammation in the CNS and disease recovery via Fas/Fas ligand-induced apoptosis of encephalitogenic T cells, and a quick resolution of inflammation in the CNS is essential to prevent permanent damage to the CNS resulting in chronic disease.     

Delta-like ligand 4 regulates central nervous system T cell accumulation during experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) T cell-mediated inflammatory demyelinating disease of the CNS that serves as a model for multiple sclerosis. Notch receptor signaling in T lymphocytes has been shown to regulate thymic selection and peripheral differentiation. In the current study, we hypothesized that Notch ligand-receptor interaction affects EAE development by regulating encephalitogenic T cell trafficking. We demonstrate that CNS-infiltrating myeloid dendritic cells, macrophages, and resident microglia expressed Delta-like ligand 4 (DLL4) after EAE induction. Treatment of mice with a DLL4-specific blocking Ab significantly inhibited the development of clinical disease induced by active priming. Furthermore, the treatment resulted in decreased CNS accumulation of mononuclear cells in the CNS. Anti-DLL4 treatment did not significantly alter development of effector cytokine expression by Ag-specific T cells. In contrast, anti-DLL4 treatment reduced T cell mRNA and functional cell surface expression of the chemokine receptors CCR2 and CCR6. Adoptive transfer of Ag-specific T cells to mice treated with anti-DLL4 resulted in decreased clinical severity and diminished Ag-specific CD4(+) T cell accumulation in the CNS. These results suggest a role for DLL4 regulation of EAE pathogenesis through modulation of T cell chemokine receptor expression and migration to the CNS.     

Inhibition of Hyaluronan Synthesis Protects against Central Nervous System (CNS) Autoimmunity and Increases CXCL12 Expression in the Inflamed CNS

Hyaluronan (HA) may have proinflammatory roles in the context of CNS autoimmunity. It accumulates in demyelinated multiple sclerosis (MS) lesions, promotes antigen presentation, and enhances T-cell activation and proliferation. HA facilitates lymphocyte binding to vessels and CNS infiltration at the CNS vascular endothelium. Furthermore, HA signals through Toll-like receptors 2 and 4 to stimulate inflammatory gene expression. We assessed the role of HA in experimental autoimmune encephalomyelitis (EAE), an animal model of MS by administration of 4-methylumbelliferone (4MU), a well established inhibitor of HA synthesis. 4MU decreased hyaluronan synthesis in vitro and in vivo. It was protective in active EAE of C57Bl/6 mice, decreased spinal inflammatory infiltrates and spinal infiltration of Th1 cells, and increased differentiation of regulatory T-cells. In adoptive transfer EAE, feeding of 4MU to donor mice significantly decreased the encephalitogenicity of lymph node cells. The transfer of proteolipid protein (PLP)-stimulated lymph node cells to 4MU-fed mice resulted in a delayed EAE onset and delayed spinal T-cell infiltration. Expression of CXCL12, an anti-inflammatory chemokine, is reduced in MS patients in CSF cells and in spinal cord tissue during EAE. Hyaluronan suppressed production of CXCL12, whereas 4MU increased spinal CXCL12 in naive animals and during neuroinflammation. Neutralization of CXCR4, the most prominent receptor of CXCL12, by administration of AMD3100 diminished the protective impact of 4MU in adoptive transfer EAE. In conclusion, hyaluronan exacerbates CNS autoimmunity, enhances encephalitogenic T-cell responses, and suppresses the protective chemokine CXCL12 in CNS tissue. Inhibition of hyaluronan synthesis with 4MU protects against an animal model of MS and may represent an important therapeutic option in MS and other neuroinflammatory diseases.