Regulation of neuroinflammation: the role of CXCL10 in lymphocyte infiltration during autoimmune encephalomyelitis

The movement of lymphocytes from the microvasculature into the central nervous system (CNS) parenchyma is an essential step in the pathogenesis of a variety of infectious and autoimmune neuroinflammatory diseases. The lymphocyte chemoattractant CXCL10 and its receptor, CXCR3, are expressed by the CNS and by CNS infiltrating lymphocytes, respectively, only in patients with ongoing CNS inflammation, suggesting an important role for these molecules in the pathogenic process. Numerous studies utilizing animal models and transgenic approaches have indeed supported a role for CXCL10 in the intraparenchymal trafficking of lymphocytes during acute CNS inflammation; however, other studies suggest that its expression is not required for the development of autoimmune forms of CNS inflammation and, in fact, that interference with CXCL10 signaling could lead to increased neuroinflammation. This review will consider the data from these studies and attempt to reconcile them through comparisons of both the neuroinflammatory models and the effects of CXCL10 in the CNS versus lymphoid tissues. Finally, it will define directions for future analyses of CXCL10 and CXCR3 in CNS inflammation so that their potential therapeutic utility can be more completely determined.     

Deletion of UCP2 in iNOS deficient mice reduces the severity of the disease during experimental autoimmune encephalomyelitis

Uncoupling protein 2 is a member of the mitochondrial anion carrier family that is widely expressed in neurons and the immune cells of humans. Deletion of Ucp2 gene in mice pre-activates the immune system leading to higher resistance toward infection and to an increased susceptibility to develop chronic inflammatory diseases as previously exemplified with the Experimental Autoimmune Encephalomyelitis (EAE), a mouse model for multiple sclerosis. Given that oxidative stress is enhanced in Ucp2-/- mice and that nitric oxide (NO) also plays a critical function in redox balance and in chronic inflammation, we generated mice deficient for both Ucp2 and iNos genes and submitted them to EAE. Mice lacking iNos gene exhibited the highest clinical score (3.4+/-0.5 p<0.05). Surprisingly, mice deficient for both genes developed milder disease with reduced immune cell infiltration, cytokines and ROS production as compared to iNos-/- mice.     

CD43 modulates severity and onset of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis characterized by infiltration of activated CD4(+) T lymphocytes into tissues of the CNS. This study investigated the role of CD43 in the induction and progression of EAE. Results demonstrate that CD43-deficient mice have reduced and delayed clinical and histological disease severity relative to CD43(+/+) mice. This reduction was characterized by decreased CD4(+) T cell infiltration of the CNS of CD43(-/-) mice but similar numbers of Ag-specific T cells in the periphery, suggesting a defect in T cell trafficking to the CNS. The absence of CD43 also affected cytokine production, as myelin oligodendrocyte glycoprotein (MOG) 35-55-specific CD43(-/-) CD4(+) T cells exhibited reduced IFN-gamma and increased IL-4 production. CD43(-/-) CD4(+) MOG-primed T cells exhibited reduced encephalitogenicity relative to CD43(+/+) cells upon adoptive transfer into naive recipients. These results suggest a role for CD43 in the differentiation and migration of MOG(35-55)-specific T cells in EAE, and identify it as a potential target for therapeutic intervention.     

Middle-age male mice have increased severity of experimental autoimmune encephalomyelitis and are unresponsive to testosterone therapy

Treatment with sex hormones is known to protect against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, little is known about how age affects the course of EAE or response to hormone treatment. This study demonstrates striking differences between middle-age vs young C57BL/6 male mice in the clinical course of EAE and response to both testosterone (T4) and estrogen (E2) hormone therapy. Unlike young males that developed an acute phase of EAE followed by a partial remission, middle-age males suffered severe chronic and unremitting EAE that was likely influenced by alterations in the distribution and function of splenic immunocytes and a significant reduction in suppressive activity of CD4+CD25+ regulatory T cells in the spleen and spinal cord. Middle-age males had reduced numbers of splenic CD4+ T cells that were generally hypoproliferative, but enhanced numbers of splenic macrophages and MHC class II-expressing cells, and increased secretion of the proinflammatory factors IFN-gamma and MCP-1. Surprisingly, middle-age males were unresponsive to the EAE-protective effects of T4 and had only a transient benefit from E2 treatment; young males were almost completely protected by both hormone treatments. T4 treatment of young males inhibited proliferation of myelin oligodendrocyte glycoprotein 35-55-specific T cells and secretion of TNF-alpha and IFN-gamma. The effects of T4 in vivo and in vitro were reversed by the androgen receptor antagonist, flutamide, indicating that the regulatory effects of T4 were mediated through the androgen receptor. These data are the first to define age-dependent differences in EAE expression and response to hormone therapy.     

实验性自身免疫性脑脊髓炎小鼠的视神经损害

目的:探讨实验性自身免疫性脑脊髓炎(EAE)的视神经损害以及将其作为视神经炎模型的可能性.方法:取MOG35-55多肽和完全弗氏佐剂制备成抗原乳剂免疫C57BL/6小鼠,并在腹腔内注射2次百日咳杆菌,建立EAE模型.在EAE疾病高峰时,观察小鼠视神经炎的发生率及病理学改变.结果:模型组12只小鼠中有10只从免疫后第12天开始陆续起病,约在第17～19天达到疾病高峰,发病率为83.3%;而对照组未出现任何神经功能受损的症状.HE染色结果显示模型组的视神经组织中有大量的炎症细胞浸润,动物视神经炎的发生率为83.3%,其炎症评分为2.1±0.8分,与对照组相比均有统计学意义.LFB染色可见模型组的视神经有明显的脱髓鞘改变,病灶内可见炎症细胞浸润;而对照组小鼠的视神经未见明显异常改变.结论:借助EAE建立视神经炎模型是可行的,这为深入探讨视神经炎的发病机制提供了理想的动物平台.     

Induction and clinical scoring of chronic-relapsing experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that commonly affects young adults. It is characterized by demyelination and glial scaring in areas disseminated in the brain and spinal cord. These lesions alter nerve conduction and induce the disabling neurological deficits that vary with the location of the demyelinated plaques in the CNS (e.g. paraparesis, paralysis, blindness, incontinence). Experimental autoimmune encephalomyelitis (EAE) is a model for MS. EAE was first induced accidentally in humans during vaccination against rabies, using viruses grown on rabbit spinal cords. Residues of spinal injected with the inactivated virus induced the CNS disease. Following these observations, a first model of EAE was described in non-human primates immunized with a CNS homogenate by Rivers and Schwenther in 1935. EAE has since been generated in a variety of species and can follow different courses depending on the species/strain and immunizing antigen used. For example, immunizing Lewis rats with myelin basic protein in emulsion with adjuvant induces an acute model of EAE, while the same antigen induces a chronic disease in guinea pigs. The EAE model described here is induced by immunizing DA rats against DA rat spinal cord in emulsion in complete Freund's adjuvant. Rats develop an ascending flaccid paralysis within 7-14 days post-immunization. Clinical signs follow a relapsing-remitting course over several weeks. Pathology shows large immune infiltrates in the CNS and demyelination plaques. Special considerations for taking care for animals with EAE are described at the end of the video.     

The extent of ultrastructural spinal cord pathology reflects disease severity in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) has been studied for decades as an animal model for human multiple sclerosis (MS). Here we performed ultrastructural analysis of corticospinal tract (CST) and motor neuron pathology in myelin oligodendrocyte glycoprotein (MOG) peptide 35-55- and MP4-induced EAE of C57BL/6 mice. Both models were clinically characterized by ascending paralysis. Our data show that CST and motor neuron pathology differentially contributed to the disease. In both MOG peptide- and MP4-induced EAE pathological changes in the CST were evident. While the MP4 model also encompassed severe motor neuron degeneration in terms of rough endoplasmic reticulum alterations, the presence of intracytoplasmic vacuoles and nuclear dissolution, both models showed motor neuron atrophy. Features of axonal damage covered mitochondrial swelling, a decrease in nearest neighbor neurofilament distance (NNND) and an increase of the oligodendroglial cytoplasm inner tongue. The extent of CST and motor neuron pathology was reflective of the severity of clinical EAE in MOG peptide- and MP4-elicited EAE. Differential targeting of CNS gray and white matter are typical features of MS pathology. The MOG peptide and MP4 model may thus be valuable tools for downstream studies of the mechanisms underlying these morphological disease correlates.     

Estrogen treatment down-regulates TNF-alpha production and reduces the severity of experimental autoimmune encephalomyelitis in cytokine knockout mice.

A shift toward Th2 cytokine production has been demonstrated during pregnancy and high dose estrogen therapy and is thought to be the primary mechanism by which estrogen suppresses the development of experimental autoimmune encephalomyelitis. However, low dose estrogen treatment is equally protective in the absence of a significant shift in cytokine production. In this study cytokine-deficient mice were treated with estrogen to determine whether a shift in Th2 cytokine production was required for the protective effects of hormone therapy. Estrogen effectively suppressed the development of experimental autoimmune encephalomyelitis in IL-4 and IL-10 knockout mice and in wild type littermate mice with a similar potency of protection. Significant disease suppression was also seen in IFN-gamma-deficient mice. The decrease in disease severity was accompanied by a concomitant reduction in the number of proinflammatory cytokine- and chemokine-producing cells in the CNS. Although there was no apparent increase in compensatory Th2 cytokine production in cytokine-deficient mice, there was a profound decrease in the frequency of TNF-alpha-producing cells in the CNS and the periphery. Therefore, we propose that one mechanism by which estrogen protects females from the development of cell-mediated autoimmunity is through a hormone-dependent regulation of TNF-alpha production.     

The thymus plays a role in oral tolerance in experimental autoimmune encephalomyelitis

The oral administration of myelin proteins has been used for the successful prevention and treatment of experimental autoimmune encephalomyelitis (EAE). We questioned whether the thymus was involved in oral tolerance. In this study, euthymic myelin basic protein (MBP) TCR transgenic mice are protected from EAE when fed MBP but are not protected when thymectomized. Similarly, in a cell transfer system, T cell responses to OVA measured in vivo were suppressed significantly only in the OVA-fed euthymic mice but not in the thymectomized mice. We observed that the absence of the thymus dramatically enhanced the Th1 response. We explored three alternatives to determine the role of the thymus in oral tolerance: 1) as a site for the induction of regulatory T cells; 2) a site for deletion of autoreactive T cells; or 3) a site for the dissemination of naive T cells. We found that Foxp3(+)CD4(+)CD25(+) T cells are increased in the periphery but not in the thymus after Ag feeding. These CD4(+)CD25(+) T cells also express glucocorticoid-induced TNFR and intracellular CTLA4 and suppress Ag-specific proliferation of CD4(+)CD25(-) cells in vitro. The thymus also plays a role in deletion of autoreactive T cells in the periphery following orally administered MBP. However, thymectomy does not result in homeostatic proliferation and the generation of memory cells in this system. Overall, the oral administration of MBP has a profound effect on systemic immune responses, mediated largely by the generation of regulatory T cells that act to prevent or suppress EAE.     

Vitamin D deficiency diminishes the severity and delays onset of experimental autoimmune encephalomyelitis

Multiple sclerosis incidence is clearly inversely related to sun exposure. This observation led to the idea that vitamin D might be responsible for this relationship. Providing super-physiologic doses of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃, suppresses an animal model of multiple sclerosis, i.e. experimental autoimmune encephalomyelitis (EAE) but causes unwanted hypercalcemia. Further, dietary calcium is needed for this activity of 1α,25-dihydroxyvitamin D₃. B10PL mice were maintained on a vitamin D-deficient diet for two generations to produce frank vitamin D deficiency. These animals showed delayed onset and reduced severity of EAE compared to control animals on the same diet and given vitamin D₃ or provided a vitamin D-containing chow diet. Thus, vitamin D deficiency interferes with the development of this autoimmune disease rather than increasing susceptibility.     

The origin and application of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a model of the neuroimmune system responding to priming with central nervous system (CNS)-restricted antigens. It is an excellent model of post-vaccinal encephalitis and a useful model of many aspects of multiple sclerosis. EAE has been established in numerous species and is induced by priming with a large number of CNS-derived antigens. As a consequence, the pathogenesis, pathology and clinical signs vary significantly between experimental protocols. As I describe in this Timeline article, the reductionist approach taken in some lines of investigation of EAE resulted in a reliance on results obtained under a narrow range of conditions. Although such studies made important contributions to our molecular understanding of inflammation, T-cell activation, and MHC restriction, they did not advance as effectively our knowledge of the polyantigenic responses that usually occur in CNS immunopathology and autoimmunity.     

Proteome analysis of spinal cord during the clinical course of monophasic experimental autoimmune encephalomyelitis

The induction of autoimmune encephalomyelitis (EAE) in Lewis rats results in a period of exacerbation followed by complete recovery. Therefore, this model is widely used for studying the evolution of multiple sclerosis. In the present investigation, differentially expressed proteins in the spinal cord of Lewis rats during the evolution of EAE were assessed using the combination of 2DE and MALDI-TOF MS. The majority of the differentially expressed proteins were identified during the acute phase of EAE, in relation to na?ve control animals. On the other hand, recovered rats presented a similar protein expression pattern in comparison with the na?ve ones. This observation can be explained, at least in part, by the intense catabolism existent in acute phase due to nervous tissue damage. In recovered rats, we have described the upregulation of proteins that are apparently involved in the recovery of damaged tissue, such as light and medium neurofilaments, glial fibrillary acidic protein, tubulins subunits, and quaking protein. These proteins are involved mainly in cell growth, myelination, and remyelination as well as in astrocyte and oligodendrocyte maturation. The present study has demonstrated that the inflammatory response, characterized by an increase of the proliferative response and infiltration of autoreactive T lymphocytes in the central nervous system, occurs simultaneously with neurodegeneration.     

Combination treatment of mice with CRx-153 (nortriptyline and desloratadine) decreases the severity of experimental autoimmune encephalomyelitis

Pro-inflammatory CD4⁺ T cell-mediated autoimmune diseases, such as multiple sclerosis, are hypothesized to be initiated and maintained by self-reactive interferon-gamma (IFN-γ) and interleukin-17 (IL-17) producing CD4⁺ T cells. Previous studies have shown moderate to significant alterations in inflammatory T cell responses and potentially treatment of autoimmune disease by administration of antihistamine or tricyclic antidepressants alone. The goal of the present study was to determine if treatment of PLP_139–151-induced relapsing–remitting experimental autoimmune encephalomyelitis (R-EAE) in SJL/J mice with a combination of two FDA approved drugs for other indications could decrease R-EAE disease. The findings show that combination treatment with desloratadine and nortriptyline decreases the mean clinical score, disease relapse frequency, and number of CD4⁺ T cells infiltrating into the CNS. In addition, combination treatment of PLP_139–151 primed mice decreases the level of IFN-γ and IL-17 secreted via a decrease in both the number of cells secreting and the amount of cytokine secreted per cell following PLP_139–151 reactivation ex vivo. This is in contrast to an increase in the level of IL-4 produced and the number of IL-4 secreting cells. The data also show that combination treatment with desloratadine and nortriptyline inhibits the production of IFN-γ and IL-17 produced by naive CD4⁺ T cells activated in the presence of Th1 cell- and Th17 cell-promoting conditions, while increasing the level of IL-4 produced by naive CD4⁺ T cells activated in the presence of Th2 cell-promoting conditions. The present findings suggest a novel method for the development of a putative autoimmune therapy.     

Fatal neurological disease in scrapie-infected mice induced for experimental autoimmune encephalomyelitis

During the years or decades of prion disease incubation, at-risk individuals are certain to encounter diverse pathological insults, such as viral and bacterial infections, autoimmune diseases, or inflammatory processes. Whether prion disease incubation time and clinical signs or otherwise the pathology of intercurrent diseases can be affected by the coinfection process is unknown. To investigate this possibility, mice infected with the scrapie agent at both high and low titers were subsequently induced for experimental autoimmune encephalomyelitis, an immune system-mediated model of central nervous system (CNS) inflammation. We show here that co-induced mice died from a progressive neurological disease long before control mice succumbed to classical scrapie. To investigate the mechanism of the co-induced syndrome, we evaluated biochemical and pathological markers of both diseases. Brain and spleen PrP(Sc) levels in the dying co-induced mice were comparable to those observed in asymptomatic scrapie-infected animals, suggesting that co-induced disease is not an accelerated form of scrapie. In contrast, inflammatory markers, such as demyelination, immune cell infiltrates, and gliosis, were markedly increased in co-induced mouse spinal cords. Activated astrocytes were especially elevated in the medulla oblongata. Furthermore, PrP(sc) depositions were found in demyelinated white matter areas in co-induced mouse spinal cords, suggesting the presence of activated infected immune cells that infiltrate into the CNS to facilitate the process of prion neuroinvasion. We hypothesize that inflammatory processes affecting the CNS may have severe clinical implications in subjects incubating prion diseases.     

Collagenase-2 deficiency or inhibition impairs experimental autoimmune encephalomyelitis in mice

Matrix metalloproteinases (MMPs) have been implicated in a variety of human diseases, including neuroimmunological disorders such as multiple sclerosis. However, the recent finding that some MMPs play paradoxical protective roles in these diseases has made necessary the detailed study of the specific function of each family member in their pathogenesis. To determine the relevance of collagenase-2 (MMP-8) in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, we have performed two different analyses involving genetic and biochemical approaches. First, we have analyzed the development of EAE in mutant mouse deficient in MMP-8, with the finding that the absence of this proteolytic enzyme is associated with a marked reduction in the clinical symptoms of EAE. We have also found that MMP-8(-/-) mice exhibit a marked reduction in central nervous system-infiltrating cells and demyelinating lesions. As a second approach, we have carried out a pharmacological inhibition of MMP-8 with a selective inhibitor against this protease (IC(50) = 0.4 nM). These studies have revealed that the administration of the MMP-8 selective inhibitor to mice with EAE also reduces the severity of the disease. Based on these findings, we conclude that MMP-8 plays an important role in EAE development and propose that this enzyme may be a novel therapeutic target in human neuro-inflammatory diseases such as multiple sclerosis.