Name: Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis (Video) | JoVE
Uploaded: 2023-05-12T14:55:00
Description: Watch this Scientific Journal Video about Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis at JoVE.c

这项工作得到了日本科学促进会的资助（资助号。JP 23K14675）。

所有小鼠实验均由顺天堂大学医学院机构动物护理和使用委员会批准（批准号290238），并按照美国国立卫生研究院动物实验指南进行。
1. 对实验的一般评论
<ol><li>在23°C±2°C，50%±10%湿度的受控无病原体条件下，以及12小时光/暗循环， 随意获取食物 和水，将小鼠饲养在动物设施中的单个笼子中。</li>
	<li>向小鼠注射不含抗原和CFA的磷酸盐缓冲盐水（PBS）?...

Immunogenicity Enhancement by Mycobacterium paratuberculosis 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>

我们展示了一种强大的替代方案，使用在含有副结核分枝杆菌10的佐剂中乳化的肽MOG35-55在C57BL / 6J小鼠中积极诱导严重的EAE。通过这种方法诱导EAE导致比CFA共同方案诱导的疾病更严重。这种差异可能是由于分枝杆菌11细胞壁中的脂质成分不同。事实上，与其他分枝杆菌不同，副结核分枝杆菌在细胞壁表面产生脂质肽抗原，而不是糖质<sup class...

实验性自身免疫性脑脊髓炎（EAE）是研究人类脱髓鞘疾病的常用模型1。EAE有几种模型：使用不同的髓磷脂肽与强效佐剂联合进行主动免疫，通过髓鞘特异性CD4 +淋巴细胞的体外转移进行被动免疫，以及自发EAE2的转基因模型。这些模型中的每一个都有特定的特征，可以研究EAE的不同方面，例如发病期，效应期或慢性期。EAE的髓鞘少突胶质细胞糖蛋白（MOG）模型是研究免疫介导的慢性神经炎症和脱髓鞘机制的良好模型，其特征是单核炎症浸润，外周白质脱髓鞘，疾病高峰后恢复减少1。
MOG-EAE是通过在完全弗氏佐剂（CFA）中用肽MOG35-55 对易感小鼠进行免疫，然后腹膜内注射百日咳毒素来诱导的。这增加了血脑屏障的通透性，并允许在外周激活的髓鞘特异性T细胞到达中枢神经系统（CNS），在那里它们将被重新激活3。CFA通过增强抗原呈递细胞的抗原摄取以及与体液和细胞介导的反应相关的细胞因子的表达，在诱导EAE中起关键作用4。这种机制主要是由于在油中乳化杀死的 结核分枝杆菌 的存在，其成分为免疫系统提供了强烈的刺激5。事实上，EAE的诱导与抗原激发期间存在的分枝杆菌数量直接相关6。
在不完全的弗氏佐剂7中添加其他被杀死的分枝杆菌，例如丁酸分枝杆菌，以及辅助组合8的作用，可以调节EAE的临床过程，从而影响结果的可重复性。 鸟分枝杆菌亚种副结核 （MAP） 是反刍动物中 Johne 病的病原体，与人类 CNS 9 的炎症性疾病有关，因为其抗原成分能够在多发性硬化症和视神经脊髓炎谱系疾病患者中引起强烈的体液和细胞介导反应9。因此，在该协议中，我们展示了一种替代且可重复的方法，通过用副结核分枝杆菌代替CFA中的结核分枝杆菌来诱导MOG-EAE。

<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

由髓磷脂少突胶质细胞糖蛋白 （MOG） 诱导的实验性自身免疫性脑脊髓炎 （EAE） 需要通过在含有灭活结 核分枝杆菌的完全弗氏佐剂 （CFA） 中乳化的 MOG 肽进行免疫接种。分枝杆菌的抗原成分激活树突状细胞，刺激T细胞产生细胞因子，通过toll样受体 促进 Th1反应。因此，抗原激发期间存在的分枝杆菌的数量和种类与EAE的发展直接相关。该方法论文提出了一种替代方案，使用含有热杀灭的 鸟分枝杆菌 亚种副 结核 菌株K-10的改良的不完全弗氏佐剂在C57BL / 6小鼠中诱导EAE。
副结核分枝杆菌是鸟分枝杆菌复合体的成员，是反刍动物中 Johne 病的病原体，已被确定为几种人类 T 细胞介导的疾病的危险因素，包括多发性硬化症。总体而言，接种副结核分枝杆菌的小鼠比接种含有结核分枝杆菌H37Ra菌株的CFA的小鼠以4mg / mL的相同剂量表现出更早的起病和更大的疾病严重程度。鸟分枝杆菌亚种副结核（MAP）菌株K-10的抗原决定簇能够在效应期诱导强烈的Th1细胞反应，其特征在于T淋巴细胞（CD4+ CD27+）、树突状细胞（CD11c+ I-A/I-E+）和单核细胞（CD11b+ CD115+）的数量显着增加）与用CFA免疫的小鼠相比。此外，副结核分枝杆菌免疫小鼠对MOG肽的增殖性T细胞反应似乎最高。在制剂中使用在含有副结核分枝杆菌的佐剂中乳化的脑炎原（例如，MOG35-55）可能是在EAE诱导阶段激活树突状细胞以启动髓鞘表位特异性CD4 + T细胞的替代和经过验证的方法。

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

C57BL / 6小鼠组（总n = 15 /组）在含有副结核分枝杆菌的乳剂中或通过CFA的常用方法用MOG35-55免疫。所有组小鼠都表现出急性单相疾病，其特征在于在14-17天观察到的单个残疾高峰，随后在接下来的10天内症状部分恢复（图1A）。用含有副结核分枝杆菌的佐剂免疫的小鼠，无论性别如何，在免疫后8至9天显示出更早的起病，并且在急?...

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

Adjuvant Activity of Mycobacterium paratuberculosis in Enhancing the Immunogenicity of Autoantigens During Experimental Autoimmune Encephalomyelitis (Video) | JoVE 

在这里，我们提出了一种替代方案，以积极诱导C57BL / 6小鼠的实验性自身免疫性脑脊髓炎，使用免疫原性表位髓鞘少突胶质细胞糖蛋白（MOG）35-55 悬浮在不完全弗氏佐剂中含有热杀灭的 鸟分枝杆菌 亚种副 结核。

副结核分枝杆菌增强实验性自身免疫性脑脊髓炎期间自身抗原免疫原的辅助活性

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

我们利用专家开发的新技术研究了几种神经系统疾病的发病机制，以改善对患有互动疾病的运动障碍患者的治疗。我们的部分研究集中在病原体的作用和新炎症性疾病（如多发性硬化症或NMOSD）的病因上，其特征是遗传和环境因素的复杂相互作用。各种病原体与这些疾病有关，尽管它们的作用尚不清楚。我们发现禽分枝杆菌细胞种类副结核的抗原成分可以在多发性硬化症患者中引起强烈的体液和细胞介导的反应。此外，我们证明了分枝杆菌通过在EAE模型中作用于肠道免疫脑轴来提供插入的自生潜力。我们将副结核分枝杆菌确定为诱导活动性EAE的有效佐剂候选物，其引起的疾病比灭活的结核分枝杆菌诱导的疾病更严重。这种差异可能是因为副结核分枝杆菌在细胞壁表面产生脂质五肽抗原而不是糖肽脂质。我们未来研究的目标之一是阐明外周神经系统和中枢神经系统免疫反应之间的串扰，并了解感染介导的神经炎症的机制。为此，我们正在开发独特的神经炎症遗传学模型。