Cytokine Control of Inflammation and Repair in the Pathology of Multiple Sclerosis

Cytokines are secreted signaling proteins that play an essential role in propagating and regulating immune responses during experimental autoimmune encephalomyelitis (EAE), the mouse model of the neurodegenerative, autoimmune disease multiple sclerosis (MS). EAE pathology is driven by a myelin-specific T cell response that is activated in the periphery and mediates the destruction of myelin upon T cell infiltration into the central nervous system (CNS). Cytokines provide cell signals both in the immune and CNS compartment, but interestingly, some have detrimental effects in the immune compartment while having beneficial effects in the CNS compartment. The complex nature of these signals will be reviewed.     

The role of immune semaphorins in multiple sclerosis

The nervous and immune systems have similar functional characteristics. Both have an intricate network of synaptic connections and an exquisite communication system that enable intercellular signal transduction. Although semaphorins were originally identified as guidance cues in neural development, accumulating evidence indicates that several semaphorins called 'immune semaphorins', such as Sema3A, 4A, 4D, 6D and 7A, are critically involved in various phases of the immune response by regulating immune cell-cell contacts or cell migration. In this review, we present recent knowledge on the functions of semaphorins and their receptors in the immune system and their potential roles in the pathogenesis of multiple sclerosis (MS), a representative CNS autoimmune disease, and its animal model, experimental autoimmune encephalomyelitis (EAE).     

Targeting VIP and PACAP receptor signalling: new therapeutic strategies in multiple sclerosis

MS (multiple sclerosis) is a chronic autoimmune and neurodegenerative pathology of the CNS (central nervous system) affecting approx. 2.5 million people worldwide. Current and emerging DMDs (disease-modifying drugs) predominantly target the immune system. These therapeutic agents slow progression and reduce severity at early stages of MS, but show little activity on the neurodegenerative component of the disease. As the latter determines permanent disability, there is a critical need to pursue alternative modalities. VIP (vasoactive intestinal peptide) and PACAP (pituitary adenylate cyclase-activating peptide) have potent anti-inflammatory and neuroprotective actions, and have shown significant activity in animal inflammatory disease models including the EAE (experimental autoimmune encephalomyelitis) MS model. Thus, their receptors have become candidate targets for inflammatory diseases. Here, we will discuss the immunomodulatory and neuroprotective actions of VIP and PACAP and their signalling pathways, and then extensively review the structure–activity relationship data and biophysical interaction studies of these peptides with their cognate receptors.     

Inhibition of calpain attenuates encephalitogenicity of MBP-specific T cells

Multiple sclerosis (MS) is a T-cell mediated autoimmune disease of the CNS, possessing both immune and neurodegenerative events that lead to disability. Adoptive transfer (AT) of myelin basic protein (MBP)-specific T cells into naïve female SJL/J mice results in a relapsing–remitting (RR) form of experimental autoimmune encephalomyelitis (EAE). Blocking the mechanisms by which MBP-specific T cells are activated before AT may help characterize the immune arm of MS and offer novel targets for therapy. One such target is calpain, which is involved in activation of T cells, migration of immune cells into the CNS, degradation of axonal and myelin proteins, and neuronal apoptosis. Thus, the hypothesis that inhibiting calpain in MBP-specific T cells would diminish their encephalitogenicity in RR-EAE mice was tested. Incubating MBP-specific T cells with the calpain inhibitor SJA6017 before AT markedly suppressed the ability of these T cells to induce clinical symptoms of RR-EAE. These reductions correlated with decreases in demyelination, inflammation, axonal damage, and loss of oligodendrocytes and neurons. Also, calpain : calpastatin ratio, production of truncated Bid, and Bax : Bcl-2 ratio, and activities of calpain and caspases, and internucleosomal DNA fragmentation were attenuated. Thus, these data suggest calpain as a promising target for treating EAE and MS.     

X-linked inhibitor of apoptosis regulates T cell effector function

To understand how the balance between pro- and anti-apoptotic signals influences effector function in the immune system, we studied the X-linked inhibitor of apoptosis (XIAP), an endogenous regulator of cellular apoptosis. Real-time PCR showed increased XIAP expression in blood of mice with experimental autoimmune encephalomyelitis, correlating with disease severity. Daily administration (10 mg/kg/day i.p.) of a 19-mer antisense oligonucleotide specific for XIAP (ASO-XIAP) abolished disease-associated XIAP mRNA and protein expression, and given from day of onset, alleviated experimental autoimmune encephalomyelitis and prevented relapses. Prophylactic treatment also reduced XIAP expression and prevented disease. Random or 5-base mismatched ASO was not inhibitory, and ASO-XIAP did not affect T cell priming. In ASO-XIAP-treated animals, infiltrating cells and inflammatory foci were dramatically reduced within the CNS. Flow cytometry showed an 88-93% reduction in T cells. The proportion of TUNEL(+) apoptotic CD4(+) T cells in the CNS was increased from <1.6 to 26% in ASO-XIAP-treated mice, and the proportion of Annexin V-positive CD4(+) T cells in the CNS increased. Neurons and oligodendrocytes were not affected; neither did apoptosis increase in liver, where XIAP knockdown also occurred. ASO-XIAP increased susceptibility of T cells to activation-induced apoptosis in vitro. Our results identify XIAP as a critical controller of apoptotic susceptibility of effector T cell function.     

IL-22RA2 associates with multiple sclerosis and macrophage effector mechanisms in experimental neuroinflammation

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the CNS. Recent advances in whole-genome screening tools have enabled discovery of several MS risk genes, the majority of which have known immune-related functions. However, disease heterogeneity and low tissue accessibility hinder functional studies of established MS risk genes. For this reason, the MS model experimental autoimmune encephalomyelitis (EAE) is often used to study neuroinflammatory disease mechanisms. In this study, we performed high-resolution linkage analysis in a rat advanced intercross line to identify an EAE-regulating quantitative trait locus, Eae29, on rat chromosome 1. Eae29 alleles from the resistant strain both conferred milder EAE and lower production of proinflammatory molecules in macrophages, as demonstrated by the congenic line, DA.PVG-Eae29 (Dc1P). The soluble IL-22R α2 gene (Il-22ra2) lies within the Eae29 locus, and its expression was reduced in Dc1P, both in activated macrophages and splenocytes from immunized rats. Moreover, a single nucleotide polymorphism located at the end of IL-22RA2 associated with MS risk in a combined Swedish and Norwegian cohort comprising 5019 subjects, displaying an odds ratio of 1.26 (p = 8.0 × 10(-4)). IL-22 and its receptors have been implicated in chronic inflammation, suggesting that IL-22RA2 regulates a central immune pathway. Through a combined approach including genetic and immunological investigation in an animal model and large-scale association studies of MS patients, we establish IL-22RA2 as an MS risk gene.     

Naringenin attenuates experimental autoimmune encephalomyelitis by protecting the intact of blood-brain barrier and controlling inflammatory cell migration

Targeting pathogenic immune cell trafficking poses an attractive opportunity to attenuate autoimmune disorders such as multiple sclerosis (MS). MS and its animal model, experimental autoimmune encephalomyelitis (EAE), are characterized by the immune cells-mediated demyelination and neurodegeneration of the central nervous system (CNS). Our previous study has proven that dietary naringenin ameliorates EAE clinical symptoms via reducing the CNS cell infiltration. The present study examined the beneficial effects of naringenin on maintaining the blood-brain barrier in EAE mice via dietary naringenin intervention. The results showed that naringenin-treated EAE mice had an intact blood-CNS barrier by increasing tight junction-associated factors and decreasing Evans Blue dye in the CNS. Naringenin decreased the accumulation and maturation of conventional dendritic cells (cDCs), CCL19, and CCR7 in the CNS. Also, naringenin blocked the chemotaxis and antigen-presenting function of cDCs that resulted in reducing T-cell secreting cytokines (IFN-γ, IL-17, and IL-6) in the spleen. Importantly, naringenin blocked pathogenic T cells infiltrated into the CNS and attenuates passive EAE. Therefore, by blocking chemokine-mediated migration of DCs and pathogenic T cells into the CNS, naringenin attenuates EAE pathogenesis and might be a potential candidate for the treatment of autoimmune diseases, such as MS and other chronic T-cell mediated autoimmune diseases.     

Cutting edge: myelin basic protein-specific cytotoxic T cell tolerance is maintained in vivo by a single dominant epitope in H-2k mice.

Multiple sclerosis (MS) is believed to be an autoimmune disease mediated by T cells specific for CNS Ags. MS lesions contain both CD4+ and CD8+ T lymphocytes. The contribution of CD4+ T cells to CNS autoimmune disease has been extensively studied in an animal model of MS, experimental autoimmune encephalomyelitis. However, little is known about the role of autoreactive CD8+ cytotoxic T cells in MS or experimental autoimmune encephalomyelitis. We demonstrate here that myelin basic protein (MBP) is processed in vivo by the MHC class I pathway leading to a MBP79-87/Kk complex. The recognition of this complex by MBP-specific cytotoxic T cells leads to a high degree of tolerance in vivo. This study is the first to show that the pool of self-reactive lymphocytes specific for MBP contain MHC class I-restricted T cells whose response is regulated in vivo by the induction of tolerance.     

The role of dorsal root ganglia activation and brain-derived neurotrophic factor in multiple sclerosis

Multiple sclerosis (MS) is characterized by focal destruction of the white matter of the brain and spinal cord. The exact mechanisms underlying the pathophysiology of the disease are unknown. Many studies have shown that MS is predominantly an autoimmune disease with an inflammatory phase followed by a demyelinating phase. Recent studies alongside current treatment strategies, including glatiramer acetate, have revealed a potential role for brain-derived neurotrophic factor (BDNF) in MS. However, the exact role of BDNF is not fully understood. We used the experimental autoimmune encephalomyelitis (EAE) model of MS in adolescent female Lewis rats to identify the role of BDNF in disease progression. Dorsal root ganglia (DRG) and spinal cords were harvested for protein and gene expression analysis every 3 days post-disease induction (pdi) up to 15 days. We show significant increases in BDNF protein and gene expression in the DRG of EAE animals at 12 dpi, which correlates with peak neurological disability. BDNF protein expression in the spinal cord was significantly increased at 12 dpi, and maintained at 15 dpi. However, there was no significant change in mRNA levels. We show evidence for the anterograde transport of BDNF protein from the DRG to the dorsal horn of the spinal cord via the dorsal roots. Increased levels of BDNF within the DRG and spinal cord in EAE may facilitate myelin repair and neuroprotection in the CNS. The anterograde transport of DRG-derived BDNF to the spinal cord may have potential implications in facilitating central myelin repair and neuroprotection.     

Elevation of Sema4A implicates Th cell skewing and the efficacy of IFN-β therapy in multiple sclerosis

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the CNS and a leading cause of lasting neurologic disabilities in young adults. Although the precise mechanism remains incompletely understood, Ag presentation and subsequent myelin-reactive CD4(+) T cell activation/differentiation are essential for the pathogenesis of MS. Although semaphorins were initially identified as axon guidance cues during neural development, several semaphorins are crucially involved in various phases of immune responses. Sema4A is one of the membrane-type class IV semaphorins, which we originally identified from the cDNA library of dendritic cell (DC). Sema4A plays critical roles in T cell activation and Th1 differentiation during the course of experimental autoimmune encephalomyelitis, an animal model of MS; however, its pathological involvement in human MS has not been determined. In this study, we report that Sema4A is increased in the sera of patients with MS. The expression of Sema4A is increased on DCs in MS patients and shed from these cells in a metalloproteinase-dependent manner. DC-derived Sema4A is not only critical for Th1 but also for Th17 cell differentiation, and MS patients with high Sema4A levels exhibit Th17 skewing. Furthermore, patients with high Sema4A levels have more severe disabilities and are unresponsive to IFN-β treatment. Taken together, our results suggest that Sema4A is involved in the pathogenesis of MS by promoting Th17 skewing.     

Production of CCL2 by central nervous system cells regulates development of murine experimental autoimmune encephalomyelitis through the recruitment of TNF- and iNOS-expressing macrophages and myeloid dendritic cells

Experimental autoimmune encephalomyelitis is a T cell-mediated demyelinating disease of the CNS that serves as a model for the human disease multiple sclerosis. Increased expression of the chemokine CCL2 in the CNS has been demonstrated to be important in the development of demyelinating disease presumably by attracting inflammatory cells. However, the mechanism of how CCL2 regulates disease pathogenesis has not been fully elucidated. Using radiation bone marrow chimeric mice we demonstrated that optimum disease was achieved when CCL2 was glia derived. Furthermore, CNS production of CCL2 resulted in the accumulation of iNOS-producing CD11b(+)CD11c(+) dendritic cells and TNF-producing macrophages important for demyelination. Lack of glial-derived CCL2 production did not influence experimental autoimmune encephalomyelitis by altering either Th1 or Th17 cells, as there were no differences in these populations in the CNS or periphery between groups. These results demonstrate that the glial-derived CCL2 is important for the attraction of TNF- and iNOS-producing dendritic cells and effector macrophages to the CNS for development of subsequent autoimmune disease.     

Adenoviral delivery of soluble VEGF receptor 1 (sFlt-1) inhibits experimental autoimmune encephalomyelitis in dark Agouti (DA) rats

Previous studies have shown that vascular endothelial growth factor (VEGF) expression is up-regulated in both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), a model for MS, and may exacerbate the disease. However, it remains unknown whether anti-VEGF modalities could serve as a potential treatment for such central nervous system (CNS) autoimmune diseases. We constructed a recombinant adenoviral vector carrying FLAG-tagged sFlt-1(1-3) (the first three extracellular domains of Flt-1, the hVEGF receptor-1). Intramuscular transfection of the recombinant adenoviral vector suppressed VEGF-induced inflammatory cell infiltration in matrigel plugs. When given intracerebrally to EAE rats, recombinant sFlt-1(1-3) adenoviral vector significantly reduced disease severity compared to untreated rats. sFlt-1(1-3) gene transfer blocked VEGF and greatly reduced the number of cells that express VEGF and ED1-positive cells in CNS in EAE rats. This study demonstrates that sFlt-1(1-3) gene transfer into the brain ameliorates the severity of EAE by inhibiting monocyte recruitment in the CNS of dark Agouti rats.     

Global and 3D Spatial Assessment of Neuroinflammation in Rodent Models of Multiple Sclerosis

Multiple Sclerosis (MS) is a progressive autoimmune inflammatory and demyelinating disease of the central nervous system (CNS). T cells play a key role in the progression of neuroinflammation in MS and also in the experimental autoimmune encephalomyelitis (EAE) animal models for the disease. A technology for quantitative and 3 dimensional (3D) spatial assessment of inflammation in this and other CNS inflammatory conditions is much needed. Here we present a procedure for 3D spatial assessment and global quantification of the development of neuroinflammation based on Optical Projection Tomography (OPT). Applying this approach to the analysis of rodent models of MS, we provide global quantitative data of the major inflammatory component as a function of the clinical course. Our data demonstrates a strong correlation between the development and progression of neuroinflammation and clinical disease in several mouse and a rat model of MS refining the information regarding the spatial dynamics of the inflammatory component in EAE. This method provides a powerful tool to investigate the effect of environmental and genetic forces and for assessing the therapeutic effects of drug therapy in animal models of MS and other neuroinflammatory/neurodegenerative disorders.     

Vra4 congenic rats with allelic differences in the class II transactivator gene display altered susceptibility to experimental autoimmune encephalomyelitis

Presentation of Ag bound to MHC class II (MHC II) molecules to CD4+ T cells is a key event in adaptive immune responses. Genetic differences in MHC II expression in the rat CNS were recently positioned to allelic variability in the CIITA gene (Mhc2ta), located within the Vra4 locus on rat chromosome 10. In this study, we have examined reciprocal Vra4-congenic strains on the DA and PVGav1 backgrounds, respectively. After experimental nerve injury the strain-specific MHC II expression on microglia was reversed in the congenic strains. Similar findings were obtained after intraparenchymal injection of IFN-gamma in the brain. Expression of MHC class II was also lower on B cells and dendritic cells from the DA.PVGav1-Vra4- congenic strain compared with DA rats after in vitro stimulation with IFN-gamma. We next explored whether Vra4 may affect the outcome of experimental autoimmune disease. In experimental autoimmune encephalomyelitis induced by immunization with myelin oligodendrocyte glycoprotein, DA.PVGav1-Vra4 rats displayed a lower disease incidence and milder disease course compared with DA, whereas both PVGav1 and PVGav1.DA-Vra4 rats were completely protected. These results demonstrate that naturally occurring allelic differences in Mhc2ta have profound effects on the quantity of MHC II expression in the CNS and on immune cells and that this genetic variability also modulates susceptibility to autoimmune neuroinflammation.     

The Synergistic Local Immunosuppressive Effects of Neural Stem Cells Expressing Indoleamine 2,3-Dioxygenase (IDO) in an Experimental Autoimmune Encephalomyelitis (EAE) Animal Model

Neurodegenerative diseases provoke robust immunological reactions in the central nervous system (CNS), which further deteriorate the neural tissue damage. We hypothesized that the expression levels of indoleamine 2,3-dioxygenase (IDO), an enzyme that has potent immune suppressive activities, in neural stem cells (NSCs) would have synergistic therapeutic effects against neurodegenerative diseases, since NSCs themselves have low IDO expression. In this study, the synergistic immune suppressive effects of rat fetal NSCs expressing IDO (rfNSCs-IDO) were validated by mixed leukocyte reaction (MLR) in vitro and an experimental autoimmune encephalomyelitis (EAE) animal model in vivo. rfNSCs-IDO showed significantly more suppressive effects on T cell proliferation in the MLR compared to control rfNSCs (rfNSCs-Cont). Importantly, IDO inhibition using 1-methyl-DL-tryptophan (1-MT), an IDO inhibitor, reversed the synergistic effects, confirming IDO-specific effects in rfNSCs-IDO. In the EAE animal model, systemic rfNSCs-IDO injections resulted in significant local immune suppression in the cervical lymph nodes and CNS, evidenced by a reduction in the number of activated T lymphocytes and an increase in regulatory T cell numbers, which induced significantly fewer clinical symptoms and faster recovery. In contrast, rfNSCs-Cont failed to reduce symptoms in the EAE animal models, although they showed local immune suppression, which was significantly less than that in rfNSCs-IDO. Taken together, IDO expression in NSCs synergistically potentiates the immune suppression activities of NSCs and could be applicable for the development of therapeutic modalities against various neurodegenerative diseases.