This work received support from a grant from the Japanese Society for the promotion of Science (grant no. JP 23K14675).

All mouse experiments were approved by the Institutional Animal Care and Use Committee of the Juntendo University School of Medicine (Approval Number 290238) and were conducted in accordance with the National Institutes of Health Guidelines for Animal Experimentation.
1. General comments on the experiment
<ol>
	<li>House the mice in individual cages in the animal facility under controlled, pathogen-free conditions at 23 &#176;C &#177; 2 &#176;C with 50% &#177; 10% humidity, and a 12 h light/dark cycle with ad libitum access to food and water.</li>
	<li>Inject the mice with phosphate-buffered saline (PBS) without antigen and CFA, and use them as negative and positive controls, respectively.</li>
</ol>
2. Preparation of mycobacterial antigens
<ol>
	<li>Grow the M. paratuberculosis K-10 strain in Middlebrook liquid medium 7H9 enriched with 10% OADC (oleic acid, albumin, dextrose, catalase), 0.005% Tween 80, and 2 mg/L of mycobactin J for 2 weeks in T25 tissue culture flasks at 37 &#176;C. Assess the colony growth regularly by visual inspection. 
	CAUTION:&#160;Handle M. paratuberculosis in a Biosafety Level 2 (BSL-2) facility.</li>
	<li>Grow the enrichment cultureina 500 mL Erlenmeyer flask with a suspension of 1.7 &#215; 105 colony-forming units (CFU)/mL in a final volume of 200 mL (same medium as step 2.1) in a shaking incubator for 1 week. 
	NOTE: As contaminating organisms may affect the results, the bacteria must be inoculated onto Middlebrook 7H10 solid media to determine colony morphology and to verify purity by conventional polymerase chain reaction (PCR) detection kits for M. paratuberculosis.</li>
	<li>Inactivate the bacterial suspensions for&#160;5-10 min at 70 &#176;C and centrifuge at 3,000 &#215; g for 10 min. Washed the weighed pellet in PBS twice, disrupt by sonication, and store at -20 &#176;C.</li>
	<li>Transfer the pellet to a sterile container and freeze it with dry ice or liquid nitrogen. Transfer immediately to a freeze-drying chamber.</li>
	<li>Freeze-dry (4 h at -50 &#176;C under vacuum) M. paratuberculosis according to the machine manual.</li>
	<li>At the end of lyophilization, remove the stainless-steel container from the freeze-dryer and transfer it to a bio-cleaning bench. Use a stainless-steel spatula to dislodge the dried MAP lumps adhering to the inner wall of the stainless-steel container and pulverize them as finely as possible.</li>
	<li>Weigh the pulverized cells using an electronic balance and manually place them in an aseptic 10 mL vial at a concentration of 10 mg/mL. 
	NOTE: The cell density was quantified as grams of dry weight per liter of sample. After 3 weeks, 1 mg (wet weight) of bacterial cell pellet contains approximately 2.5 &#215; 108 CFU.</li>
</ol>
3. Preparation of MOG&#160;35-55 emulsion
<ol>
	<li>Grind the contents of one vial of dried M. paratuberculosis (10 mg) to a fine powder with a mortar and pestle and add 10 mL to incomplete Freund&#39;s adjuvant to obtain a 10 mg/mL stock solution. Store at -4 &#176;C.</li>
	<li>Prior to immunization, dilute the stock solution to a final concentration of 4 mg/mL.</li>
	<li>Dilute the peptide MOG35-55 in ddH2O to a final concentration of 2 mg/mL and store at -20 &#176;C.</li>
	<li>Using a homogenizer, mix the MOG35-55 solution (2 mg/mL) with an equal volume of adjuvant (4 mg/mL) from step 3.2 in a 5 mL tube until a thick emulsion is formed. After every 10 s of mixing, place the solution on ice for 20 s, and spin the tube to recover all the solution.</li>
	<li>Transfer the emulsion into a 1 mL syringe, remove all air, and add a 27 G needle. The emulsion is now ready for injection. 
	NOTE. Since some loss of the viscous emulsion of MOG35-55 occurs during preparation, it is best to prepare 1.5 times the amount needed.</li>
</ol>
4. Animal immunization
<ol>
	<li>Anesthetize the animals with inhalation of isoflurane to minimize stress on the animals.</li>
	<li>Inject the emulsion (200 &#181;L containing 200 &#181;g/mouse of MOG35-55)&#160;subcutaneously into the lower back.</li>
	<li>Administer a dose of pertussis toxin (100 &#181;L containing 200 ng/mouse) intraperitoneally on days 0 and 2 after immunization. 
	&#8203;CAUTION: Pertussis toxin has a multiple suppressive effect on the immune system; avoid ingestion and contact with eyes and skin.</li>
</ol>
5. Clinical assessment
<ol>
	<li>Monitor the mice daily for weight and clinical signs. Use the following scoring system: 0 = no clinical signs; 1 = flaccid tail; 2 = impairment of the righting reflex and weakness of the hind limbs; 3 = complete paralysis of the hind limbs; 4 = complete paralysis of the hind limbs with partial paralysis of the forelimbs; 5 = moribund. 
	NOTE: Upon the initial observation of clinical signs, animals are provided with increased access to water and food. Rodent chow is softened and moistened, then placed on the cage floor from the food hopper. In the case of dehydrated animals, subcutaneous fluid supplementation is administered, or a rodent chow slurry is given via oral gavage. If a mouse necessitates this type of assistance for over 3 days, euthanasia will be conducted.</li>
	<li>To avoid or minimize animal pain and distress, use the following human endpoints: body weight loss greater than 20%, clinical score &#8805;4.0, absence of the righting reflex at score 3, and when the animal is unable to access feed or water for 24 h. 
	NOTE: The mice were euthanized on day 30 after EAE induction by intraperitoneal injections of pentobarbital (&#8805;150 mg/kg).</li>
</ol>

Immunogenicity Enhancement by Mycobacterium paratuberculosis in Autoimmune Encephalomyelitis

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	<li>Bittner, S., Afzali, A. M., Wiendl, H., Meuth, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+oligodendrocyte+glycoprotein+(MOG35-55)+induced+experimental+autoimmune+encephalomyelitis+(EAE)+in+C57BL/6+mice.">Myelin oligodendrocyte glycoprotein (MOG35-55) induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice.</a> Journal of Visualized Experiments. (86), e51275 (2014).</li><li>Constantinescu, C. S., Farooqi, N., O'Brien, K., Gran, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+encephalomyelitis+(EAE)+as+a+model+for+multiple+sclerosis+(MS).">Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS).</a> British Journal of Pharmacology. 164 (4), 1079-1106 (2011).</li><li>Lu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pertussis+toxin+induces+angiogenesis+in+brain+microvascular+endothelial+cells.">Pertussis toxin induces angiogenesis in brain microvascular endothelial cells.</a> Journal of Neuroscience Research. 86 (12), 2624-2640 (2008).</li><li>Awate, S., Babiuk, L. A., Mutwiri, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+action+of+adjuvants.">Mechanisms of action of adjuvants.</a> Frontiers in Immunology. 4, 114 (2013).</li><li>Kubota, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adjuvant+activity+of+Mycobacteria-derived+mycolic+acids.">Adjuvant activity of Mycobacteria-derived mycolic acids.</a> Heliyon. 6 (5), e04064 (2020).</li><li>Nicolo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+in+the+adjuvant+modulates+the+balance+of+Th+immune+response+to+self-antigen+of+the+CNS+without+influencing+a+&amp;#34;core&amp;#34;+repertoire+of+specific+T+cells.">Mycobacterium tuberculosis in the adjuvant modulates the balance of Th immune response to self-antigen of the CNS without influencing a &amp;#34;core&amp;#34; repertoire of specific T cells.</a> International Immunology. 18 (2), 363-374 (2006).</li><li>O'Connor, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adjuvant+immunotherapy+of+experimental+autoimmune+encephalomyelitis:+immature+myeloid+cells+expressing+CXCL10+and+CXCL16+attract+CXCR3+CXCR6++and+myelin-specific+T+cells+to+the+draining+lymph+nodes+rather+than+the+central+nervous+system.">Adjuvant immunotherapy of experimental autoimmune encephalomyelitis: immature myeloid cells expressing CXCL10 and CXCL16 attract CXCR3+CXCR6+ and myelin-specific T cells to the draining lymph nodes rather than the central nervous system.</a> Journal of Immunology. 188 (5), 2093-2101 (2012).</li><li>Libbey, J. E., Fujinami, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+encephalomyelitis+as+a+testing+paradigm+for+adjuvants+and+vaccines.">Experimental autoimmune encephalomyelitis as a testing paradigm for adjuvants and vaccines.</a> Vaccine. 29 (17), 3356-3362 (2011).</li><li>Cossu, D., Yokoyama, K., Hattori, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conflicting+role+of+Mycobacterium+species+in+multiple+sclerosis.">Conflicting role of Mycobacterium species in multiple sclerosis.</a> Frontiers in Neurology. 8, 216 (2017).</li><li>Cossu, D., Yokoyama, K., Sakanishi, T., Momotani, E., Hattori, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adjuvant+and+antigenic+properties+of+Mycobacterium+avium+subsp.+paratuberculosis+on+experimental+autoimmune+encephalomyelitis.">Adjuvant and antigenic properties of Mycobacterium avium subsp. paratuberculosis on experimental autoimmune encephalomyelitis.</a> Journal of Neuroimmunology. 330, 174-177 (2019).</li><li>Biet, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopentapeptide+induces+a+strong+host+humoral+response+and+distinguishes+Mycobacterium+avium+subsp.+paratuberculosis+from+M.+avium+subsp.+avium.">Lipopentapeptide induces a strong host humoral response and distinguishes Mycobacterium avium subsp. paratuberculosis from M. avium subsp. avium.</a> Vaccine. 26 (2), 257-268 (2008).</li><li>Cossu, D., Yokoyama, K., Tomizawa, Y., Momotani, E., Hattori, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+humoral+immunity+to+mycobacterial+antigens+in+Japanese+patients+affected+by+inflammatory+demyelinating+diseases+of+the+central+nervous+system.">Altered humoral immunity to mycobacterial antigens in Japanese patients affected by inflammatory demyelinating diseases of the central nervous system.</a> Scientific Reports. 7 (1), 3179 (2017).</li><li>Cossu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mucosal+immune+response+induced+by+oral+administration+of+heat-killed+Mycobacterium+avium+subsp.+paratuberculosis+exacerbates+EAE.">A mucosal immune response induced by oral administration of heat-killed Mycobacterium avium subsp. paratuberculosis exacerbates EAE.</a> Journal of Neuroimmunology. 352, 577477 (2021).</li><li>Cossu, D., Yokoyama, K., Sakanishi, T., Sechi, L. A., Hattori, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+Calmette-Guerin+Tokyo-172+vaccine+provides+age-related+neuroprotection+in+actively+induced+and+spontaneous+experimental+autoimmune+encephalomyelitis+models.">Bacillus Calmette-Guerin Tokyo-172 vaccine provides age-related neuroprotection in actively induced and spontaneous experimental autoimmune encephalomyelitis models.</a> Clinical and Experimental Immunology. 6, (2023).</li></ol>

The authors have no conflicts of interest to disclose.

We demonstrated a robust alternative protocol to actively induce severe EAE in C57BL/6J mice using the peptide MOG35-55 emulsified in an adjuvant containing M. paratuberculosis10. The induction of EAE by this method resulted in a more severe disease than that induced by the common protocol with CFA. This difference could be due to the different lipidic components in the cell wall of the mycobacteria11. In fact, unlike other mycobacteria, M. paratuber...

Experimental autoimmune encephalomyelitis (EAE) is a common model for the study of human demyelinating disorders1. There are several models of EAE: active immunization using different myelin peptides in combination with potent adjuvants, passive immunization by in vitro transfer of myelin-specific CD4+ lymphocytes, and transgenic models of spontaneous EAE2. Each of these models has specific features that allow different aspects of EAE to be studied, such as the onset, effector phase, or chronic phase. The myelin oligodendrocyte glycoprotein (MOG) model of EAE is a good model to study the immune-mediated mechanisms of chronic neuroinflammation and demyelination, as it is characterized by mononuclear inflammatory infiltration, demyelination in peripheral white matter, and reduced recovery after the disease peak1.
MOG-EAE is induced by immunization of susceptible mice with the peptide MOG35-55 in complete Freund&#39;s adjuvant (CFA), followed by an intraperitoneal injection of pertussis toxin. This increases the permeability of the blood-brain barrier and allows myelin-specific T-cells activated in the periphery to reach the central nervous system (CNS), where they will be reactivated3. CFA plays a key role in the induction of EAE by enhancing the antigen uptake by antigen-presenting cells and the expression of cytokines related to humoral- and cell-mediated responses4. This mechanism is mainly due to the presence of killed Mycobacterium tuberculosis emulsified in oil, the components of which provide a strong stimulus for the immune system5. In fact, the induction of EAE is directly related to the amount of mycobacterium present during the antigenic challenge6.
The addition of other killed mycobacteria, such as Mycobacterium butyricum, to incomplete Freund&#39;s adjuvant7, as well as the effect of adjuvant combinations8, can modulate the clinical course of EAE and consequently influence the reproducibility of the results. Mycobacterium avium subspecies paratuberculosis (MAP), the etiologic agent of Johne&#39;s disease in ruminants, has been associated with inflammatory disorders of the human CNS9, as its antigenic components are capable of eliciting a strong humoral- and cell-mediated response in patients with multiple sclerosis and neuromyelitis optica spectrum disorder9. Therefore, in this protocol, we show an alternative and reproducible method to induce MOG-EAE by replacing M. tuberculosis in CFA with M. paratuberculosis.

<table><tbody><tr><td>anti-mouse CD115 antibody</td><td>Biolegend, USA</td><td>135505</td><td>for cytofluorimetry 1:1,000</td></tr><tr><td>anti-mouse CD11b antibody</td><td>Biolegend, USA</td><td>101215</td><td>for cytofluorimetry 1:1,000</td></tr><tr><td>anti-mouse CD11c antibody</td><td>Biolegend, USA</td><td>117313</td><td>for cytofluorimetry 1:1,000</td></tr><tr><td>anti-mouse CD16/32&amp;nbsp; antibody</td><td>Biolegend, USA</td><td>101302</td><td>for cytofluorimetry 1:1,000</td></tr><tr><td>anti-mouse CD4&amp;nbsp; antibody</td><td>Biolegend, USA</td><td>116004</td><td>for cytofluorimetry 1:1,000</td></tr><tr><td>anti-mouse CD8a&amp;nbsp; antibody</td><td>Biolegend, USA</td><td>100753</td><td>for cytofluorimetry 1:1,000</td></tr><tr><td>anti-mouse I-A/I-E antibody</td><td>Biolegend, USA</td><td>107635</td><td>for cytofluorimetry 1:1,000</td></tr><tr><td>anti-mouse Ly-6C&amp;nbsp; antibody</td><td>Biolegend, USA</td><td>128023</td><td>for cytofluorimetry 1:1,000</td></tr><tr><td>BBL Middlebrook OADC Enrichment</td><td>Thermo Fisher Scientific, USA</td><td>BD 211886</td><td>for isolation and cultivation of mycobacteria</td></tr><tr><td>C57BL/6J mice</td><td>Charles River Laboratory, Japan</td><td></td><td>3 weeks old, male and female</td></tr><tr><td>FBS 10279-106</td><td>Gibco Life Techologies, USA</td><td>42F9155K</td><td>for cell culture, warm at 37 &amp;deg;C before use</td></tr><tr><td>Freeze Dryer machine</td><td>Eyela, Tokyo, Japan</td><td>FDU-1200</td><td>for bacteria lyophilization</td></tr><tr><td>incomplete e Freund&amp;rsquo;s adjuvant</td><td>Difco Laboratories, MD, USA</td><td>263810</td><td>for use in adjuvant</td></tr><tr><td>Middlebrook 7H9 Broth</td><td>Difco Laboratories, MD, USA</td><td>90003-876</td><td>help in the growth of Mycobacteria</td></tr><tr><td>Mycobacterium avium subsp. paratuberculosis K-10</td><td>ATCC, USA</td><td>BAA-968</td><td>bacteria from bovine origin</td></tr><tr><td>Mycobacterium tuberculosis H37 Ra, Desiccated</td><td>BD Biosciences, USA</td><td>743-26880-EA</td><td>for use in adjuvant</td></tr><tr><td>Mycobactin J</td><td>Allied Laboratory, MO, USA</td><td></td><td>growth promoter</td></tr><tr><td>Myelin Oligodendrocyte Glycolipid (MOG) 35-55</td><td>AnaSpec, USA</td><td>AS-60130-10</td><td>encephalotigenic peptide</td></tr><tr><td>Ovalbumin (257-264)</td><td>Sigma-Aldrich, USA</td><td>S7951-1MG</td><td>negative control antigen&amp;nbsp; for proliferative assay</td></tr><tr><td>pertussis toxin solution</td><td>Fujifilm Wako, Osaka Japan</td><td>168-22471</td><td>From gram-negative bacteria Bordetella pertussi, increases blood-brain barrier permeability</td></tr><tr><td>Polytron homogenizer PT 3100</td><td>Kinematica</td><td></td><td>for mixing the antigen with the adjuvant</td></tr><tr><td>RPMI 1640 with L-glutamine</td><td>Gibco Life Techologies, USA</td><td>11875093</td><td>For cell culture</td></tr><tr><td>Thymidine, [Methyl-&lt;sup&gt;3&lt;/sup&gt;H], in 2% ethanol, 1 mCi</td><td>PerkinElmer, Waltham, MA, USA</td><td>NET027W001MC</td><td>for proliferation assay, use (1 &amp;mu;Ci/well)</td></tr><tr><td>Zombie NIR Fixable Viability Kit</td><td>Biolegend, USA</td><td>423105</td><td>&amp;nbsp;cytofluorimetry, for cell viability</td></tr></tbody></table>

adjuvant activity of mycobacterium paratuberculosis in enhancing the immunogenicity of autoantigens during experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in complete Freund&#39;s adjuvant (CFA) containing inactivated Mycobacterium tuberculosis. The antigenic components of the mycobacterium activate dendritic cells to stimulate T-cells to produce cytokines that promote the Th1 response via toll-like receptors. Therefore, the amount and species of mycobacteria present during the antigenic challenge are directly related to the development of EAE. This methods paper presents an alternative protocol to induce EAE in C57BL/6 mice using a modified incomplete Freund&#39;s adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis strain K-10.
M. paratuberculosis, a member of the Mycobacterium avium complex, is the causative agent of Johne&#39;s disease in ruminants and has been identified as a risk factor for several human T-cell-mediated disorders, including multiple sclerosis. Overall, mice immunized with Mycobacterium paratuberculosis showed earlier onset and greater disease severity than mice immunized with CFA containing the strain of M. tuberculosis H37Ra at the same doses of 4 mg/mL. The antigenic determinants of Mycobacterium avium subspecies paratuberculosis (MAP)&#160;strain K-10 were able to induce a strong Th1 cellular response during the effector phase, characterized by significantly higher numbers of T-lymphocytes (CD4+ CD27+), dendritic cells (CD11c+ I-A/I-E+), and monocytes (CD11b+ CD115+) in the spleen compared to mice immunized with CFA. Furthermore, the proliferative T-cell response to the MOG peptide appeared to be highest in M. paratuberculosis-immunized mice. The use of an encephalitogen (e.g., MOG35-55) emulsified in an adjuvant containing M. paratuberculosis in the formulation may be an alternative and validated method to activate dendritic cells for priming myelin epitope-specific CD4+ T-cells during the induction phase of EAE.

Groups of C57BL/6 mice (total n = 15/group) were immunized with MOG35-55 in an emulsion containing M. paratuberculosis or by the common method with CFA. All groups of mice manifested an acute monophasic disease characterized by a single peak of disability observed at 14-17 days, followed by a partial recovery of symptoms over the next 10 days (Figure 1A). Mice immunized with the adjuvant containing M. paratuberculosis, irrespectiv...

Watch this Scientific Journal Video about Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis at JoVE.c

Author Spotlight: Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis  

Here, we present an alternative protocol to actively induce experimental autoimmune encephalomyelitis in C57BL/6 mice, using the immunogenic epitope myelin oligodendrocyte glycoprotein (MOG)35-55 suspended in incomplete Freund's adjuvant containing the heat-killed Mycobacterium avium subspecies paratuberculosis.

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) requires immunization by a MOG peptide emulsified in ...

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Immunology and Infection

2.0K Views.  Juntendo University. We bestudeerden de pathogenese van verschillende neurologische ziekten met nieuwe technieken ontwikkeld door onze experts om de behandeling te verbeteren voor patiënten met bewegingsstoornissen die interactieve ziekten hebben. Een deel van ons onderzoek richt zich op de rol van pathogenen en de etiologie van nieuwe ontstekingsziekten zoals multiple sclerose of NMOSD, die worden gekenmerkt door een complex samenspel van genetische en omgevingsfactoren. Verschillende pathogenen zijn in verband...

Video: Auteur in de schijnwerpers: adjuvante activiteit van Mycobacterium paratuberculosis bij het verbeteren van de immunogeniciteit van auto-antigenen tijdens experimentele auto-immuunencefalomyelitis  

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

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.

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) ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely used as a model to identify new drug targets for better patient treatment. MS is characterized by several different disease courses: relapsing-remitting MS (RRMS), primary progressive MS (PPMS), secondary progressive MS (SPMS), and a rare progressive-relapsing form of MS (PRMS). Although animal models do not accurately mimic all of these contrasting human disease phenotypes, there are EAE models that reflect some of the different clinical manifestations of MS. For example, myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6J mice mimics human PPMS, while myelin proteolipid protein (PLP)-induced EAE in SJL/J mice resembles RRMS. Other autoantigens, such as myelin basic protein (MBP), and a number of different mouse strains are also used to study EAE. To induce disease in these autoantigen-immunization EAE models, a water-in-oil emulsion is prepared and injected subcutaneously. The majority of EAE models also require an injection of pertussis toxin for the disease to develop. For consistent and reproducible EAE induction, a detailed protocol to prepare the reagents to produce antigen/adjuvant emulsions is necessary. The method described here takes advantage of a standardized method to generate water-in-oil emulsions. It is simple and fast and uses a shaking homogenizer instead of syringes to prepare quality-controlled emulsions.

To induce experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, mice are immunized with a water-in-oil emulsion containing an autoantigen and complete Freund's adjuvant. While several protocols exist for the preparation of these emulsions, a rapid, simple, and standardized homogenization protocol for emulsion preparation is presented here.

Experimental autoimmune encephalomyelitis (EAE) shares similar immunological and clinical features with multiple sclerosis (MS), and is therefore widely ...

Investigating Humoral Response in Autoimmune Demyelination

Quantification of Autoreactive Antibodies in Mice upon Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological and molecular levels. EAE is induced by immunizing experimental animals via subcutaneous injection of short myelin peptides together with specific adjuvants to boost the immune response. Like the human counterpart, EAE mice develop demyelinating lesions, immune cell infiltration into the central nervous system (CNS), glia activation and neuronal injury. A consistent body of evidence also supports a mechanistic role for B cell dysfunction in the etiology of both MS and EAE. B cells can serve as antigen-presenting cells as well as a primary source of pro-inflammatory cytokines and autoantibodies. In EAE, antibodies are generated against the myelin peptides that were employed to induce the disease. Such autoantibodies have been shown to mediate either myelin loss or pathogenic T cell reactivation into the CNS. This article describes an efficient ELISA-based protocol to quantify autoantibodies in the serum of C57BL/6J mice immunized with the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) peptide. The proposed method serves as a powerful tool to investigate the specificity and magnitude of the aberrant humoral response in the context of autoimmune demyelination.

Author Spotlight: Novel Assay for Studying B-Cell Responses in Multiple Sclerosis Research

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which shares with the human disease a robust humoral autoimmune response. Here, we report a simple and flexible ELISA protocol to quantify autoantibodies in the serum of EAE immunized mice.

Experimental autoimmune encephalomyelitis (EAE) is a disease model that recapitulates the autoimmune disorder multiple sclerosis (MS) at histopathological ...

Induction and Evaluation of Experimental Autoimmune Encephalomyelitis in Mouse

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

Author Spotlight: Creating a Versatile Experimental Autoimmune Encephalomyelitis Model Relevant for Both Male and Female Mice 

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different ...

Neuroscience

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and axonal damage. Currently, there are no measures to cure MS completely, but multiple disease-modifying therapies (DMT) are available to control and mitigate disease progression. There are significant similarities between the CNS pathological features of experimental autoimmune encephalomyelitis (EAE) and MS patients. EAE has been widely used as a representative model to determine MS drugs' efficacy and explore the development of new therapies for MS disease. Active induction of EAE in mice has a stable and reproducible effect and is particularly suitable for studying the effects of drugs or genes on autoimmune neuroinflammation. The method of immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG35-55) and the daily assessment of disease symptoms using a clinical scoring system is mainly shared. Given the complex etiology of MS with diverse clinical manifestations, the existing clinical scoring system can't satisfy the assessment of disease treatment. To avoid the shortcomings of a single intervention, new indicators to assess EAE based on clinical manifestations of anxiety-like moods and osteoporosis in MS patients are created to provide a more comprehensive assessment of MS treatment.

The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and ...

Biology

Generation of Experimental Autoimmune Encephalomyelitis in a Mouse Model

Source: Tietz, S. M. et al. <a href="https://app.jove.com/v/59249">Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo.</a> J. Vis. Exp.&#160; (2019)
This video demonstrates the method of inducing experimental autoimmune encephalomyelitis (EAE) by injecting myelin-derived peptides with an adjuvant to activate autoreactive T cells, which migrate to the brain. Pertussis toxin is administered to increase blood-brain barrier permeability, allowing these T cells and other immune cells to enter the brain. These immune cells cause demyelination, leading to impaired nerve transmission and development of EAE.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. EAE ...

JoVE Encyclopedia of Experiments

Neuropathology

Cerebrovascular Diseases

Preparing an Antigen-Adjuvant Emulsion to Induce Autoimmune Encephalomyelitis

Source: B&#228;ckstr&#246;m, B. T. <a href="https://app.jove.com/v/64634">A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental Autoimmune Encephalomyelitis.</a> J. Vis. Exp. (2022)
This video demonstrates an efficient homogenization technique to generate an antigen-adjuvant emulsion to induce experimental autoimmune encephalomyelitis (EAE) in a murine model. The emulsion contains an autoantigen &#8212; myelin oligodendrocyte glycoprotein (MOG) &#8212; in the water phase, surrounded by a continuous oil phase comprising an inactivated pathogenic bacteria. The emulsion ensures a sustained release of antigens for a prolonged immune response.

Source: B&#228;ckstr&#246;m, B. T. A Rapid, Simple, and Standardized Homogenization Method to Prepare Antigen/Adjuvant Emulsions for Inducing Experimental ...

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis

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