Name: a strategy for the study of il-9-producing lymphoid cells in the nippostrongylus brasiliensis infection model
Uploaded: 2023-03-03T14:30:00
Description: Watch this Scientific Journal Video about A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model at JoVE.com

作者希望感谢何塞·路易斯·拉莫斯-巴尔德拉斯的技术支持。这项工作得到了CONACYT（FORDECYT-PRONACE-303027）对PLL的以下资助的支持。OM-P和EO-M获得了CONACYT的研究金（分别为736162和481437）。MCM-M 获得了 CONACYT 的奖学金（墨西哥 2022 年 （3））。

这里描述的所有动物实验都得到了墨西哥国立自治大学细胞生理学研究所动物处理内部委员会（CICUAL）的批准。
注意：整个协议的流程图如图 1 所示。
1. 老鼠的住房
<ol><li>使用8-10周龄的雌性或雄性小鼠组，在12小时光照/黑暗循环中饲养在具有恒定温度和湿度的动物设施中， 随意获取水和 食物。 注意：该协?...

<ol>
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要全面了解肠道寄生虫-宿主相互作用和对蠕虫感染的免疫反应，需要鉴定和分析不同的细胞群和效应分子，这些细胞群和效应分子是诱导组织重塑和蠕虫排出的关键。土壤传播的蠕虫感染是全世界发展中国家的一个大问题。然而，直到最近，还没有允许分析受这种感染影响的主要器官小肠中存在的稀有细胞群的方案5。该协议涵盖了先前描述的方法，这些方法允许在 巴西奈瑟...

钩虫是肠道寄生虫，感染全世界约7亿人，主要在不发达国家的热带地区。 十二指肠钩瘤 和美洲钩虫（人类最常见的 钩虫寄生虫）的高强度感染会导致贫血和蛋白质缺乏，从而导致生长和智力发育延迟1。 美洲猪笼草 和啮齿动物寄生虫巴西 圆线虫 在其宿主中诱导原型2型免疫反应，并且在其生命周期中具有相似性。因此， 巴西猪笼草 小鼠感染是人类钩虫感染最常用的模型。3期（L3） 巴西猪笼草 感染性幼虫在感染后的最初几个小时内从皮肤移动到肺部。一旦进入肺部，它们就会变成L4并沿着气管向上迁移，被吞咽，通过胃，并在4-5天内到达肠道成为成虫（L5）。在肠道中，L5蠕虫产卵，这些卵在粪便中排泄以重新启动寄生虫生命周期2。
巴西猪笼草诱导的免疫应答的特征是几种 2 型细胞因子增加，包括 IL-4、IL-5、IL-9、IL-10 和 IL-13，以及嗜酸性粒细胞增多、嗜碱性粒细胞增多、杯状细胞和肥大细胞增生，以及 IgG1 和 IgE 生成增加。大多数试图识别和定义巴西奈瑟菌感染时引发的免疫反应的研究都集中在IL-4或IL-13在该模型3中的作用上。然而，表达IL-9的细胞的鉴定和表征以及这种细胞因子的功能在很大程度上被忽视了，直到Licona-Limón等人发表了第一项研究，证明IL-9在针对巴西猪笼草的免疫反应中的关键作用。本研究使用报告小鼠将T细胞（主要是辅助性T细胞9）和2型先天淋巴细胞（ILC2）描述为感染时表达IL-9的主要细胞亚群4。
从蠕虫感染的肺部分离和表征免疫细胞是可行的，并且已被广泛报道3，4。然而，由于固有的组织重塑和粘液的产生，在受感染的肠道中这样做被证明是一个技术挑战，直到最近Ferrer-Font等人5的发表。该小组概述了一种方案，用于分离和分析来自 Heligmosomoides polygyrus感染的鼠肠的免疫亚群的单细胞悬浮液。基于此，我们现在已经标准化了从 巴西猪笼草 感染肠道中分离和细胞术分析表达IL-9的淋巴细胞的方案。此外，我们已经建立了来自整个感染过程中不同细胞来源和解剖位置的IL-9动力学。
表征参与这种感染的不同细胞群对于更广泛地了解对寄生虫的免疫反应及其与宿主的相互作用至关重要。这个全面的方案提供了一条明确的途径，可以在感兴趣的疾病阶段从所需器官中分离和分析产生IL-9的细胞，从而可以显着提高有关这些细胞在 巴西猪笼草 感染和寄生虫感染中的作用的知识。

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	<tbody>
		<tr>
			<td>ACK buffer</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Attune Nxt cytometer</td>
			<td>Thermofisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B220</td>
			<td>Biolegend</td>
			<td>103204</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>Biolegend</td>
			<td>101204</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11c&#160;</td>
			<td>Biolegend</td>
			<td>117304</td>
			<td></td>
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		<tr>
			<td>CD19&#160;</td>
			<td>Biolegend</td>
			<td>115504</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>Biolegend</td>
			<td>100404</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4 (BV421)</td>
			<td>Biolegend</td>
			<td>100443</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45.2</td>
			<td>Biolegend</td>
			<td>109846</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8&#160;</td>
			<td>Biolegend</td>
			<td>100703</td>
			<td></td>
		</tr>
		<tr>
			<td>CD90.2</td>
			<td>Biolegend</td>
			<td>105314</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088866001</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Invitrogen</td>
			<td>18068015</td>
			<td>Specific activity: &#8805;10 000 units/mg&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>Facs ARIA II sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Melody cell sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-Block</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc&#949;RI</td>
			<td>eBioscience</td>
			<td>13589885</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>FlowJo</td>
			<td></td>
			<td>Flow cytometry analysis data software</td>
		</tr>
		<tr>
			<td>Gr-1</td>
			<td>Tonbo</td>
			<td>305931</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution (HBSS)</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IL-9</td>
			<td>biolegend</td>
			<td>514103</td>
			<td></td>
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		<tr>
			<td>NK1.1&#160;</td>
			<td>Biolegend</td>
			<td>108704</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh</td>
			<td>&#160;&#8206; lba</td>
			<td>B07HYHHX5V</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep Density Gradient Medium</td>
			<td>Sigma</td>
			<td>D1556</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline&#160;</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Siglec F&#160;</td>
			<td>Biolegend</td>
			<td>155512</td>
			<td></td>
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		<tr>
			<td>Streptavidin</td>
			<td>Biolegend</td>
			<td>405206</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR-&#946;&#160;</td>
			<td>Biolegend</td>
			<td>109203</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR-&#946; (PE/Cy7)</td>
			<td>Biolegend</td>
			<td>109222</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR-&#947;&#948;&#160;</td>
			<td>Biolegend</td>
			<td>118103</td>
			<td></td>
		</tr>
		<tr>
			<td>Ter119</td>
			<td>Biolegend</td>
			<td>116204</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricine buffer&#160;</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie Aqua Fixable Viability Dye</td>
			<td>Biolegend</td>
			<td>423101</td>
			<td></td>
		</tr>
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a strategy for the study of il-9-producing lymphoid cells in the nippostrongylus brasiliensis infection model

IL-9是一种多效性细胞因子，与各种过程相关，包括抗肿瘤免疫，过敏病理诱导和对蠕虫感染的免疫反应，其中它在寄生虫的排出中起重要作用。在 巴西黑圆线虫 感染的小鼠模型中，IL-9主要由肺、小肠和引流淋巴结中发现的CD4 + T淋巴细胞和先天淋巴细胞产生。鉴于IL-9细胞内染色所涉及的技术困难，以及在感染时从小肠分离造血细胞的复杂性，迫切需要一个全面但直接的方案来分析IL-9在该模型中不同淋巴和非淋巴组织中的表达。此处描述的方案概述了CD4 + T细胞和先天淋巴样细胞在整个感染过程中在肺和小肠（ 巴西奈瑟菌靶向的主要器官）以及纵隔和肠系膜淋巴结中产生的IL-9的动力学。此外，它还详细说明了感染所需的幼虫数量，具体取决于感兴趣的细胞类型和器官。该协议旨在通过提供专注于 巴西猪笼草 感染模型中感兴趣的特定细胞、器官和疾病阶段的机会，协助测定的标准化以节省时间和资源。

小鼠皮下注射200 L3期 巴西猪笼草 幼虫，或用PBS进行假对照。调整了该协议中使用的幼虫数量，以便从肺，淋巴组织和小肠中分离出活细胞，这与以前的报告不同，以前的报告使用更高的蠕虫负荷来检测淋巴组织和肺部中的细胞仅4。在感染后第 0、4、7 和 10 天采集肺、纵隔淋巴结、肠系膜淋巴结和小肠，用于淋巴细胞分离和 IL-9 生成人群的特征。在两个或多个独立实验中?...

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A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

表达IL-9的T和ILC2细胞在 巴西猪笼草 感染期间被诱导，但由于它们的低频率和差异动力学，它们的表征在感染的肠道中在很大程度上被忽视了。该协议描述了从不同靶器官中分离这些细胞，并通过不同感染阶段的流式细胞术 确认 其身份。

巴西黑线虫感染模型中产生IL-9的淋巴细胞研究策略

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response ...

This protocol allows for the isolation and characterization of lymphoid cells expressing IL-9 in N.brasiliensis non-lymphoid infected tissues including the infected cut which recently was technically invisible.This protocol also provides an alternative to analyze populations of cells from peripheral tissues previously unappreciated in infection due to the intrinsic difficulties of isolating cells from these anatomical sites.This method allows the study of difficult-to-isolate populations from N.brasiliensis infected cut and could be applied to other helminthic infections, broadening our understanding of hookworm infections in humans.Demonstrating the procedure will be Ofelia Mu

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JoVE Journal

Immunology and Infection

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

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