Name: induction of experimental autoimmune encephalomyelitis in mice and evaluation of the disease-dependent distribution of immune cells in various tissues
Uploaded: 2016-05-08T14:00:00
Description: Watch this Scientific Journal Video about Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues at JoVE

This work was supported by the Else Kr&#246;ner-Fresenius Foundation (EKFS) Research Training Group Translational Research Innovation - Pharma (TRIP) and by the "Landesoffensive zur Entwicklung wissenschaftlich-&#246;konomischer Exzellenz (LOEWE), Schwerpunkt: Anwendungsorientierte Arzneimittelforschung" of the State of Hesse.

ETHICS STATEMENT: Our experimental procedures are approved by the Ethics Committee of the Regierungspr&#228;sidium Darmstadt (Germany) and confirm to National and European regulations. All efforts were made to minimize animal suffering and reduce the number of animals used.
1. EAE Model
<ol>
	<li>Induction of the EAE model
	<ol>
		<li>Use 10- to 13-week-old female 129S4/SvJae&#215;C57BL/6 mice for the induction of EAE.</li>
		<li>Give the mice a subcutaneous injection, into the upper and low...

The EAE model described here has received the most attention as a model of MS and is routinely used in testing therapeutic strategies for MS32. The mouse disease exhibits many clinical and histological features of MS and is caused by the induction of autoimmunity to neuronal antigens. The sensitization to myelin antigens is associated with blood brain barrier dysfunction and thereby, immune cell infiltration into the CNS. Our findings show that immune cells increase transiently in the lymph nodes in the acute ...

Multiple sclerosis (MS) and its corresponding animal model, experimental autoimmune encephalomyelitis (EAE), show autoimmune neuroinflammation changes in the central nervous system (CNS). Early active MS and EAE lesions are characterized by the presence of infiltrated immune cells. The etiology of MS remains unknown, but is widely considered to involve the destruction of myelin mediated by autoreactive T cells. These autoreactive T cells secrete pro-inflammatory cytokines and chemokines which attract other immune cells such as B cells, monocytes and neutrophils from the circulation. Monocytes differentiate into macrophages. Interferon gamma (IFN&#947;) secreted by autoreactive T cell polarizes the macrophages into pro-inflammatory macrophages. The pro-inflammatory macrophages release cytokines and reactive oxygen species that promote apoptosis in oligodendrocytes. The death of the oligodendrocytes leads to demyelination. Furthermore, B cells differentiate into plasma cells and release autoantibodies against the myelin sheath, ultimately resulting in degradation of myelin. The loss of myelin leads to degradation of axons and neurons and thereby to the formation of lesion sites in the CNS which represent the main characteristic of MS1. In the periphery, T cells and B cells are activated in the lymph nodes, they proliferate in the spleen and migrate through the circulation into the central nervous system. Monocytes and neutrophils proliferate in the bone marrow and also migrate through the circulation into the central nervous system.
Leukocyte extravasation from bone marrow, spleen and lymph nodes into the blood or from the bloodstream into the CNS is a multistep process that depends on several factors, including molecular interactions between leukocytes and endothelium mediated by chemokines and chemokine receptors. The production of chemokines by various cell types can be induced during the immune reaction by cytokines like tumor necrosis factor-&#945; (TNF&#945;), IFN&#947; and interleukin-6 (IL-6), which subsequently recruits immune cells to the site of inflammation2,3. Immune cells present a subset of chemokine receptors on their surface, depending on cell type and migration pathway to the inflammatory site. Thus, CXCR2, CCR1 and CXCR1 are expressed on mature neutrophils in bone marrow and blood4, and binding of its ligands, CXCL2, CCL5 or CXCL6, respectively, activates neutrophils and promotes their adhesion to the endothelium and subsequently, the migration of the cells into tissues5-9. CCL2 and CCL20 attract monocytes and Th1/Th17 cells10, which express CCR211 and CCR612, respectively. CCR1 and CCR5, expressed by different cell types, including T cells, monocytes and macrophages13, bind CCL3, CCL5 and CCL7 and are upregulated during MS14. CXCR3 is expressed on T cells and binds CCL9, CCL10 and CCL1115.
One main strategy in MS treatment is the depletion of immune cells or the prevention of immune cell infiltration into the CNS. Therefore, the blockade of specific chemokine receptors has been investigated in EAE. Antagonism or genetic deletion of CCR116, CCR217, CCR718 or CXCR219 reduces EAE pathology, whereas antagonism or genetic deletion of CCR120, CCR520 or CXCR321 did not reduce the pathology. Hence, the expression of specific chemokine receptors on leukocytes is crucial for the infiltration of the latter into the CNS and dictates the course of EAE.
The depletion of immune cells is an effective treatment strategy for MS patients, because infiltrated immune cells release cytokines, such as TNF&#945;, IL-6 and IL-1&#946;, which, in turn, promote the inflammatory process or the degradation of neurons22. Furthermore, auto-reactive Th1 cells release IFN&#947;, which in turn stimulates macrophages to release TNF&#945;, IL-1&#946; and IL-23.
This manuscript describes the induction of EAE, the determination of the immune cell distribution and the cytokine levels (mRNA) in various tissues in EAE mice. Cells were isolated at different time points during the disease course to provide a time-dependent overview of the inflammatory processes which finally lead to lesion formation in the CNS.

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induction of experimental autoimmune encephalomyelitis in mice and evaluation of the disease-dependent distribution of immune cells in various tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

Figure 1 gives a schematic overview of the different methods described in this article. 1) Mice receive an injection of MOG35-55 antigen and develop initial clinical symptoms after 10.7 &#177; 0.3 days28. A representative disease course of EAE mice is shown in Figure 1. 2) Various tissues (spleen, lymph nodes, lumbar spinal cord) and blood are extracted from control and EAE mice at different time poi...

Watch this Scientific Journal Video about Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues at JoVE

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Research

JoVE Journal

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