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, ...

Immunogenicity Enhancement by &lt;em&gt;Mycobacterium paratuberculosis&lt;/em&gt; in Autoimmune Encephalomyelitis

<ol>
	<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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160; antibody</td>
			<td>Biolegend, USA</td>
			<td>101302</td>
			<td>for cytofluorimetry 1:1,000</td>
		</tr>
		<tr>
			<td>anti-mouse CD4&#160; antibody</td>
			<td>Biolegend, USA</td>
			<td>116004</td>
			<td>for cytofluorimetry 1:1,000</td>
		</tr>
		<tr>
			<td>anti-mouse CD8a&#160; 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&#160; 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 &#176;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&#8217;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&#160; 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-3H], in 2% ethanol, 1 mCi</td>
			<td>PerkinElmer, Waltham, MA, USA</td>
			<td>NET027W001MC</td>
			<td>for proliferation assay, use (1 &#956;Ci/well)</td>
		</tr>
		<tr>
			<td>Zombie NIR Fixable Viability Kit</td>
			<td>Biolegend, USA</td>
			<td>423105</td>
			<td>&#160;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.

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

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

We studied the pathogenesis of several neurological diseases with new techniques developed by our experts to improve treatment for patient with movement disorders who have interactive diseases. Part of our research focuses on the role of pathogens and the etiology of new inflammatory diseases such as multiple sclerosis or NMOSD, which are characterized by a complex interplay of genetic and environmental factors. Various pathogens have been associated with these diseases, although their role is unclear.We found that antigenic components of Mycobacterium avian cell species paratuberculosis can elicit a strong humoral and cell-mediated response in patient with multiple sclerosis. Furthermore, we demonstrated that Mycobacteria provide inset autogenic potential by acting on the gut immune brain axis in the EAE model. We identified Mycobacterium paratuberculosis as a potent adjuvant candidate in the induction of active EAE, which resulted in a more severe disease than that induced by inactivated Mycobacterium tuberculosis.This difference could be because Mycobacterium paratuberculosis produces a lipo pentapeptide antigen on the cell wall surface instead of glycopeptide lipids. One of the goals of our future research is to elucidate the crosstalk between the immune response of the peripheral and central nervous system, and to understand the mechanism of infection-mediated neuroinflammation. For this, we were developing unique genetics model of neuroinflammations.

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

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.

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

adjuvant-activity-mycobacterium-paratuberculosis-enhancing

Research

JoVE Journal

Immunology and Infection

Mycobacterial Antigen Preparation

Begin by growing mycobacterium paratuberculosis K-10 strain in T25 flasks containing enriched medium for two weeks at 37 degrees Celsius. Assess the colony growth regularly through visual inspection. Grow 200 milliliters of the enrichment culture in a 500 milliliter erlenmeyer flask in a shaking incubator for one week.Place the bacterial suspensions at 70 degrees Celsius for five to 10 minutes to inactivate them and centrifuge them at 3000G for 10 minutes. Wash the pellets in 20 milliliters of PBS twice and centrifuge the suspension. Transfer the pellet to a sterile container and freeze it with liquid nitrogen.Transfer the frozen pellet to a lyophilizer chamber immediately. After lyophilization transfer it to a bio cleaning bench. Using a stainless steel spatula dislodge the dried MAP lumps adhering to the inner walls of the container and pulverize them to a fine powder.Weigh the pulverized cells and manually place them in an aseptic 10 milliliter vial.

Preparation and Injection of Myelin Oligodendrocyte Glycoprotein (MOG35-55) Emulsion

Preparation and Injection of Myelin Oligodendrocyte Glycoprotein (MOG35-55) Emulsion  

Begin by grinding 10 milligrams of freeze-dried Mycobacterium paratuberculosis to a fine powder using a mortar and pestle. Add five milliliters of Incomplete Freund's adjuvant to obtain a 10 milligram per milliliter stock solution. Store the stock at minus four degrees Celsius.Before immunization, dilute the stock solution. Then, dilute the MOG 35-55 peptide to two milligrams per milliliter and store it at minus 20 degrees Celsius. Mix equal volumes of the MOG 35-55 with the adjuvant in a five milliliter tube with a homogenizer until a thick emulsion is formed.After every 10 seconds of mixing, place the solution on ice for 20 seconds and spin the tube to recover all the solution. Transfer the emulsion into a one milliliter syringe, ensuring it is free of air bubbles. Then, fit it with a 27-gauge needle.Subcutaneously, inject 200 microliters of the emulsion into the lower back of an anesthetized mouse. Then, intraperitoneally inject a dose of pertussis toxin on day zero and two after immunization. All mice manifested an acute monophasic disease, characterized by a single peak of disability observed at 14 to 17 days, followed by a partial recovery of symptoms over the next 10 days.Mice immunized with M.paratuberculosis showed an earlier onset after immunization and greater severity in the acute phase. Both groups demonstrated similar body weights. Proliferation assay with titrated thymidine incorporation performed on the EAE mice spleen cells showed a strong proliferative response to the peptide MOG 35-55, but not to ovalbumin.Cytofluorometric analysis showed increased T-lymphocytes, dendritic cells, and monocytes in the spleen of M.paratuberculosis-immunized EAE mice compared to CFA-immunized mice. Hematoxylin and and eosin tissue analysis revealed typical paravascular and meningeal mononuclear inflammatory infiltration in the brain and spinal cord.

1.2K Views.  Juntendo University.  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. 

Immunogenicity Enhancement by Mycobacterium paratuberculosis in Autoimmune Encephalomyelitis

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