巨噬细胞内细胞内生长的定量化是评价利什曼原虫寄生虫毒力的快速可靠方法
Summary
所有致病性的利什曼原虫物种在其脊椎动物宿主的巨噬细胞内栖息和复制。在这里, 我们提出了一个协议, 以感染小鼠骨髓源性巨噬细胞的文化与利什曼原虫, 然后精确定量的细胞内生长动力学。该方法对于研究影响寄主病原体相互作用和利什曼原虫毒力的个体因素非常有用。
Abstract
利什曼原虫的生命周期, 利什曼病的致病剂, 分别在昆虫和脊椎动物寄主 promastigote 和 amastigote 阶段交替。虽然利什曼病的致病症状可能有很大差异, 从良性皮肤病变到高度致命的内脏疾病的形式取决于感染的物种, 所有的利什曼原虫物种驻留在宿主巨噬细胞在脊椎动物阶段他们的生命周期。因此,利什曼原虫传染性直接关系到它在巨噬细胞内 parasitophorous 泡 (PVs) 中侵入、生存和复制的能力。因此, 评估寄生虫复制 intracellularly 的能力是确定毒力的可靠方法。研究利什曼病的发展使用动物模型是费时, 繁琐, 往往是困难的, 特别是与 pathogenically 重要的内脏形式。我们这里描述了一个方法来遵循的细胞内发育的利什曼原虫骨髓源性巨噬细胞 (BMMs)。细胞内寄生虫的数量是确定的24小时间隔为 72-96 h 继感染后。这种方法可以可靠地确定各种遗传因素对利什曼原虫毒力的影响。举例来说, 我们展示了单个等位基因删除利什曼原虫线粒体铁转运基因 (LMIT1) 如何削弱利什曼原虫 amazonensis突变株LMIT1/ΔLmit1在 BMMs 内生长的能力,反映了与野生型相比, 毒力的急剧减少。这种检测还允许精确控制实验条件, 可以单独操作, 分析各种因素 (营养素、活性氧种类、等等) 对寄主-病原体相互作用的影响。因此, 适当的执行和量化的法钢公司感染研究提供了一个非侵入性, 快速, 经济, 安全和可靠的替代传统动物模型研究。
Introduction
利什曼病是指由利什曼原虫属的原虫寄生种类引起的广泛的人类疾病。目前约有1200万人感染了全球范围内的利什曼原虫, 3.5亿多名患者面临风险。疾病病理学依赖于利什曼原虫物种和寄主因素, 症状不同于无害的自我愈合皮肤病变和致命的 visceralizing 形式。如果未经治疗, 内脏利什曼病是致命的, 只有在疟疾后才会成为感染了原生动物寄生虫的最致命的人类疾病1。尽管疾病病理和症状存在广泛的差异, 但所有的利什曼原虫物种分别在昆虫和脊椎动物寄主的 promastigote 和 amastigote 阶段之间有 digenic 的生命周期交替。在脊椎动物体内,利什曼原虫目标宿主巨噬细胞侵入, 并诱导 parasitophorous 液泡 (PVs) 的形成, 酸性隔层具有高度剧毒 amastigote 形态复制的 phagolysosomes 特性。Amastigotes 持续在宿主组织中的慢性感染, 可以传递到未感染的白蛉, 完成传输周期。因此, 在人类疾病发展的背景下, amastigotes 是最重要的利什曼原虫生命周期表单2。调查 amastigotes 如何在巨噬细胞 PVs 中复制对于了解利什曼原虫毒力3,4,5,6,7和新的有效疗法的发展。
我们在这里描述了我们的实验室经常使用的方法来研究在骨髓源巨噬细胞 (BMMs) 中的利什曼原虫感染和复制, 这涉及到定量评估细胞内的利什曼原虫的数量随着时间的推移。该过程包括从小鼠骨髓中获取单核细胞和分化为培养的巨噬细胞,体外感染与感染型 (metacyclic 前鞭毛体或 amastigotes)的利什曼原虫和量化的每隔24小时胞内寄生虫的数量在感染后 72-96 小时内。本试验已在实验室中使用, 以确定几种环境因素和寄生虫基因的影响, 包括鉴定铁在促进L. amazonensis毒力方面的关键作用, 进一步验证了都安小鼠病变发育研究6,8,9,10,11,12,13,14,15.由于所有致病性的利什曼原虫物种都在宿主巨噬细胞内建立了它们的复制利基, 这种检测方法可普遍用于所有利什曼原虫物种的毒力测定。
执行法钢公司感染可以分析单个细胞层面上的寄主寄生虫相互作用, 从而更广泛地了解利什曼原虫寄生虫如何与他们的首选宿主微环境、巨噬细胞的 PVs 相互作用。巨噬细胞感染化验已成功使用多个组16,17,18,19,20,21,22 , 探索宿主巨噬细胞和利什曼原虫特定基因的功能, 以及它们在细胞内感染的复杂相互作用中的潜在参与。法钢公司感染允许将寄生虫生长量量化为对影响细胞内生存的宿主因素的影响的读出, 如杀菌剂一氧化氮生产、产生活性氧种类和其他不利条件在溶酶体样 PVs23中遇到。巨噬细胞感染的检测也被用来识别潜在的抗 leishmanial 药物治疗发展的线索13,24。
法钢公司感染的体外性质比其他评估利什曼原虫毒力的方法具有多种优势。然而, 以前的几项研究表明, 随着时间的推移, 细胞内寄生虫存活的机制并没有量化感染率为20,21,24。此外, 许多研究侧重于以下的体内感染随着时间的推移, 通过测量皮肤病变大小和其他生理症状只是间接相关的寄生虫复制25,26,27.体内感染是一种严格的评估寄生虫毒力的方法, 但仅基于都安肿胀的病变大小测量往往是不够的, 因为它们反映了感染组织的炎症反应, 而不是寄生虫的绝对数量。因此, 都安病变的发展化验必须遵循量化的寄生虫负荷的感染组织, 这一程序需要冗长的限制稀释化验28。此外,体内研究通常涉及在不同时间点牺牲多个动物以提取感兴趣的组织6,8,9,10,11,13. 相比之下, 大量的 BMMs 可以从一只动物身上获得, 这些细胞可以在允许在不同时间点对感染进行评估的条件下进行镀膜。此外, 与体内研究相比, 执行体外法钢公司感染可以更好地控制实验条件。量化的巨噬细胞感染与寄生虫本身允许精确控制感染的多样性 (语言) 和文化条件。对这些因素的精细控制是识别离散细胞通路特征和了解它们对感染过程的影响的关键。
考虑到这些优势, 对于研究利什曼原虫毒力的少数群体迄今充分利用细胞内复制的定量评估, 这一点令人吃惊。在本文中, 我们讨论了可能妨碍更广泛地利用此检测的常见缺陷, 并提供了一步一步的协议, 以促进其适当的实施。考虑到它的精确性和通用性, 我们在这里描述的法钢公司感染检测方法不仅可以用来探索影响利什曼原虫毒力的寄主病原体相互作用, 而且还能研究在体内复制的其他微生物。巨噬细胞29。重要的是, 这种检测也可以作为一种快速、经济的临床前筛选方法, 用于抗 leishmanial 药物的开发。
Protocol
Representative Results
Discussion
上述法钢公司感染试验所产生的定量数据, 使调查人员能够在相对较短的时间内获得感染率和可靠地测定毒力特性的变化 (最多2周, 比2体内实验所需的月份)。这种方法依赖于 DNA 特异染料 DAPI, 专门染色巨噬细胞和寄生虫细胞核, 并允许快速识别和量化感染细胞。相比之下, 其他污渍, 如姬姆萨绑定到大量不同的细胞分量, 具有不同的强度, 复杂的视觉分析37。使用 DAPI 允许识?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究院资助 RO1 AI067979 NWA。
YK 是霍华德休斯医学院/马里兰大学帕克分校的本科生奖学金获得者。
Materials
| 6 well cell culture plate | Cellstar | 657160 | |
| Adenine | Acros Organics | AC147440250 | |
| Aerosol Barrier Pipet Tips (100-1000 μL) | Fisherbrand | 02-707-404 | |
| Aerosol Barrier Pipet Tips (20-200 μL) | Fisherbrand | 02-707-430 | |
| Aerosol Barrier Pipet Tips (2-20 μL) | Fisherbrand | 02-707-432 | |
| Bard-Parker Rib-Back Carbon Steel Surgical Blade #10 | Aspen Surgical | 371110 | |
| BD Luer-Lok Tip 10 mL Syringe | Becton Dickinson (BD) | 309604 | |
| BD Precisionglide Needle, 25G | Becton Dickinson (BD) | 305124 | |
| Cell Culture Dish 35 mm x 10 mm | Cellstar | 627 160 | |
| Cell Culture Flask | Cellstar | 660175 | |
| Cover Glasses: 12 mm circles | Fisherbrand | 12-545-80 | |
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Invitrogen | D1306 | |
| D-Biotin | J.T. Baker | C272-00 | |
| EDTA | Sigma Aldrich | EDS | |
| Ethyl alcohol 200 proof | Pharmco-AAPER | 111000200 | |
| Falcon 100 mm x 15 mm non-TC-treated polystyrene Petri dish | Corning | 351029 | |
| Fetal Bovine Serum | Seradigm | 1500-500 | |
| Ficoll400 | Sigma Aldrich | F8016 | |
| Fluorescence Microscope | Nikon | E200 | |
| Goat anti-mouse IgG Texas red | Invitrogen | T-862 | |
| Goat anti-rabbit IgG AlexaFluor488 | Invitrogen | A-11034 | |
| Hemin | Tokyo Chemical Industry Co. LTD | H0008 | |
| HEPES (1M) | Gibco | 15630-080 | |
| Isoton II Diluent | Beckman Coulter | 8546719 | |
| L-Glutamine | Gemini | 400-106 | |
| Medium 199 (10X) | Gibco | 11825-015 | |
| Na pyruvate (100 mM) | Gibco | 11360-070 | |
| Paraformaldehyde | Alfa Aesar | 43368 | |
| Penicillin/Streptomycin | Gemini | 400-109 | |
| Phosphate Buffered Saline (-/-) | ThermoFisher | 14200166 | |
| Polypropyline conical Centrifuge Tubes 15 mL | Cellstar | 188 271 | |
| Polypropyline conical Centrifuge Tubes 50 mL | Cellstar | 227 261 | |
| ProLong Gold antifade reagent | ThermoFisher | P36930 | |
| Rat anti-mouse Lamp-1 antibody | Developmental Studies Hybridoma Bank | 1D4B | |
| Recombinant Human M-CSF | PeproTech | 300-25 | |
| Reichert Bright-Line Hemocytometer | Hausser Scientific | 1492 | |
| RPMI Medium 1640 (1X) | Gibco | 11875-093 | |
| Triton X-100 Surfactant | Millipore Sigma | TX1568-1 | |
| Trypan Blue | Sigma Aldrich | T8154 | |
| Delicate Scissors, 4 1/2" | VWR | 82027-582 | |
| Dissecting Forceps, Fine Tip | VWR | 82027-386 | |
| Microscope Slides | VWR | 16004-368 | |
| Z1 Coulter Particle Counter, Dual Threshold | Beckman Coulter | 6605699 |
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