从老鼠皮肤及引流淋巴结髓细胞分离皮下继免疫减毒活<em>疟原虫</em>孢子
Summary
We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.
Abstract
当疟原虫的子孢子阶段接种到哺乳动物宿主的通过蚊子叮咬皮肤疟疾感染开始。高度能动的寄生虫不仅到达肝脏侵入肝细胞转化为红细胞感染形式。它也迁移到皮肤和引流注射部位，在那里它可以被识别和居民和/或招募骨髓细胞退化近侧淋巴结。活体成像报道在皮肤发明亮荧光的赖氨酸GFP阳性白细胞和在引流淋巴结的子孢子和的CD11c +细胞之间的相互作用的早期募集。在这里，我们提出了一个有效的步骤来恢复，识别和枚举招募到小鼠皮肤和引流淋巴结继小鼠模型免疫剂量子孢子的皮内注射骨髓细胞亚群。表型特征采用多参数流式细胞仪提供一个可靠的检测，以评估疟原虫感染的炎症反应在早期的动态细胞变化。
Introduction
疟疾是最致命的传染病在世界上，造成每年超过五十万的人。感染疟原虫 ，该疾病的致病因子，开始由预红细胞（PE）的阶段。在这个阶段中，注入到由阴按蚊宿主皮肤的子孢子达到通过血流肝脏和分化内肝细胞成感染红血细胞，并导致疾病的症状的寄生虫的形式。
疟原虫的PE阶段代表了抗疟疾疫苗特权的目标。事实上，对住这些阶段减毒疫苗，如辐射衰减孢子（RAS），基因逮捕寄生虫（GAP）或药物预防和孢子（CPS）已经证明他们保护啮齿类和人类宿主1-9的能力。在啮齿动物模型，大多数疫苗研究是利用进行静脉免疫，这是在保护效力方面的黄金标准。然而，皮肤阶段的描述，并在引发保护皮肤相关引流淋巴结（DLN）的重要性，已经改变了我们的PE阶段的感知，并强调喷射的皮内途径的重要性。 P的活体成像注入啮齿动物的皮肤疟原虫子孢子已经表明，只有〜25％的接种的到达经由血流肝脏。剩余的〜75％的基DLN（〜15％）和皮肤（〜50％）10,11，其中一小部分可以皮肤细胞12,13内变换和仍然存活数周之间分配。此外，随后的研究描述了皮内免疫后建立有效的保护性免疫的主要发生在皮肤-DLN，其中寄生虫特异性CD8 + T细胞被激活的地方，并且仅在脾或肝DLNS 14,15轻微。
而大多数研究集中于在建立保护性免疫应答的牵连的效应细胞的表征，较少被知道关于注射进皮肤活的减毒的寄生虫的命运，特别是它们的先天免疫系统的相互作用。特别是，涉及在寄生虫抗原摄取，加工和呈递至CD8 + T细胞的抗原呈递细胞的表征是至关重要的，因为知道PE抗原采集既可以在皮肤和DLN隔间发生。上一页活体成像的研究以下感染蚊子叮咬16，而在DLN 10,17观察孢子和树突状细胞之间的相互作用早在肌肤明亮描述荧光赖氨酸-GFP阳性细胞的早期涌入。最近，已经报道，在由蚊子皮肤接种孢子同时增加树突和调节性T细胞的运动中的皮肤小鼠中，而在DLN 18观察抗原呈递细胞的减少数量。
我们的目的是确定和量化更精确地招募在皮肤和相应DLN以及那些以下免疫剂量的RAS 19的皮内注射与寄生虫相互作用的白细胞亚群。在这方面，我们分离出两种组织的骨髓细胞（CD45 +细胞CD11b +）和由多=参数流特征的感兴趣的亚群术。在利什曼原虫皮肤感染20的早期阶段中描述的免疫应答相一致，主主机响应于子孢子注射由随后的炎性单核细胞（CD45 +细胞CD11b中性粒细胞（CD45 +细胞CD11b + Ly6G +的Ly6C INT）的一个连续招募+ Ly6G –的Ly6C +），这些differenti的基础上确定的在Ly6G和的Ly6C表面标记的人表达。
我们在这里描述用于从小鼠皮肤中分离的骨髓细胞和以下免疫从感染蚊唾液腺提取剂量的RAS的皮内注射DLN的协议。可重复的皮内注射和组织处理的量化感染的组织内浸润的细胞群的表型变化的关键步骤。下面详述的方法提供了一种可靠的测定法来评估皮肤和DLN炎性响应于疟原虫寄生虫和可扩展到各种实验系统。
Protocol
Representative Results
Discussion
在使用全子孢子疟疾疫苗的人类大规模疫苗接种的角度来看，要克服的主要挑战之一是开发优化的路线和寄生虫的给药方法，以确保成功的免疫和保护24,25。在人类中，由减毒活寄生虫（LAP）介导的保护效力的评估具有以下自然蚊子被执行叮咬2，以及皮内，皮下25,26和IV免疫27。如在啮齿类动物28和非人灵长类25报道，LAP的IV施用相比，上述路线诱导?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢帕特里夏巴达西，凡妮莎Lagal和萨宾Thiberge为批判性阅读，伊琳娜·多布雷斯库和萨宾Thiberge的帮助在小鼠皮肤拍照和Pauline Formaglio教学中的子孢子蠕动体内成像。我们还要感谢马立克Szatanik和中心的生产和按蚊（CEPIA – 巴斯德研究所）感染蚊子饲养。这项研究是由安盛研究基金和基金从科特迪瓦了Laboratoire卓越“新发传染性疾病的综合生物学”（批准号。ANR-10的LabX-62-IBEID）。
Materials
| Ketamine: Imalgene® 1000 | Merial | – | – |
| Xylazine: Rompun® 2% | Bayer | – | – |
| NanoFil syringe + 35 gauge needle | World Precision Instruments | – | – |
| Omnican® 50 Insulin syringe 0,5 ml/50 I.U. | B. Braun Medical | 9151125 | – |
| MultiwellTM 6 well tissue culture plate – Flat Bottom | BD Falcon | 353046 | – |
| 70 µm cell strainer | BD Falcon | 352350 | – |
| 2 ml syringe | Terumo | SS-02S | – |
| BLUE MAXTM 15ml Polypropylene conical tube | BD Falcon | 352097 | – |
| BLUE MAXTM 50ml Polypropylene conical tube | BD Falcon | 352098 | – |
| 5ml Polystyrene Round-Bottom Tube with 35µm Cell-Strainer Cap | BD Falcon | 352235 | – |
| DPBS 1X Cacl2- and MgCl2-free | Life Technologies | 14190-094 | – |
| DMEM 1X + GlutaMAXTM | Life Technologies | 31966-021 | – |
| Collagenase from Clostridium histolyticum, Type IV 0.5-5.0 FALGPA units/mg solid | Sigma-Aldrich | C5138 | 400 U/ml |
| Deoxyribonuclease I from bovine pancreas, type IV | Sigma-Aldrich | D5025 | 50 µg/ml |
| EDTA disodium salt | Sigma-Aldrich | E-5134 | 10mM or 2.5 mM |
| FBS | Biowest | S1810-500 | – |
| HEPES buffer solution (1M) | Gibco | 15630-056 | 25 mM |
| Trypan blue Stain (0,4%) | Life Technologies | 15250-061 | Dilution 1:10 |
| Anti-mouse CD16/CD32 (2.4G2 clone) | BD Biosciences | 553142 | 10µg/ml final (1:50) |
| DAPI FluoroPureTM grade | Life Technologies | D21490 | 1µg/ml final |
| Anti-mouse CD45 (30-F11 clone) | BD Biosciences | 559864 | Dilution 1:200 |
| Anti-mouse CD11b (M1/70 clone) | BD Biosciences | 557657 | Dilution 1:400 |
| Anti-mouse CD8α (5H10 clone) | Life Technologies | MCD0830 | Dilution 1:100 |
| Female C57BL/6JRj mice (7-week-old) | Janvier Laboratories | – | – |
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