果蝇、果蝇细胞免疫应答的评估
Summary
该方案描述了一种在成虫体内吞噬试验的方法, 以量化吞噬细胞的识别和微生物感染的清除。
Abstract
在所有动物中, 先天免疫提供了对广泛的病原体的即时和强大的防御。体液和细胞免疫反应是先天免疫的主要分支, 许多调节这些反应的因素在无脊椎动物和哺乳动物之间在进化上是保守的。吞噬作用是细胞先天免疫的核心成分, 由免疫系统的专门血细胞进行。果蝇果蝇已成为研究整个动物吞噬的分子机制和生理影响的有力遗传模型。在这里, 我们演示了一种基于注射的体内吞噬试验, 以量化亲精细胞、血细胞对颗粒的吸收和破坏。该程序允许研究人员精确控制颗粒浓度和剂量, 从而能够在短时间内获得高度可重复的结果。该实验是定量的, 易于执行, 并可用于筛选影响病原体识别、吸收和清除的宿主因子。
Introduction
天生的免疫防御是抵御病原微生物的第一道防线。这些反应可以在功能上分为体液和细胞先天免疫, 这两个反应都是由细菌线编码模式识别受体 (Prr) 介导的, 感知病原体相关的分子模式 (Pampp)1。许多先天免疫的信号通路和效应机制在哺乳动物和无脊椎动物中得到了保护, 如线虫、线虫和果蝇、果蝇2。果蝇已成为研究宿主防御传染性微生物的有力系统 3.五味子在基因上是可追踪的, 在实验室里容易和廉价饲养, 并且有很短的世代时间。此外, 果蝇对一系列微生物表现出高效的防御能力, 从而能够检查宿主对病毒、细菌、真菌或寄生虫病原体的免疫力。
果蝇免疫学家历来利用前瞻性基因筛选、基因组范围 rna 介导的昆虫细胞系干扰 (rnai) 筛选以及先前存在的突变苍蝇株来检查先天免疫–从而导致识别和表征几种进化保守的体液免疫途径4,5,6,7,8。可以说, 体液先天免疫反应是果蝇免疫防御的最佳特征。感染后, 体液反应导致产生和系统释放的抗菌肽 (AMP) 分子进入血淋巴, 在昆虫的血液当量。Amp 是由高度保守的收费和 Imd 信号通路产生的。Toll 通路与哺乳动物 TLRXIL-1R 受体信号转导同源, Imd 通路与哺乳动物肿瘤坏死因子α信号转相同源。在食人鱼中,恐惧症是由革兰氏杆菌诱导的, 霉菌、真菌和食子蛋白 x viru6、9、10和 imd 信号是由革兰氏菌诱导的. ,12岁。
细胞免疫, 由侵入性病原体的包封、黑色素化和吞噬组成, 由称为血细胞13的专门血细胞进行。果蝇血细胞有三类: 晶体细胞、层状细胞和浆细胞13。晶体细胞占幼虫循环血细胞的 5%, 它释放异丙氧酶 (proPO) 酶, 导致病原体和宿主组织在伤口部位的黑色素化。在健康的胚胎或幼虫中通常找不到的细胞是包封异物的粘附细胞。这些细胞是在产后或寄生黄蜂卵沉积在幼虫体内时诱导的。吞噬性浆细胞占幼虫循环血流细胞的 95%, 在成人所有剩余血细胞中发挥着作用, 在发育过程中对组织重塑起着重要作用, 值得注意的是, 吞噬细胞是嗜铬菌细胞免疫的主要效应细胞。
吞噬作用是先天免疫防御的一条关键线;破坏宿主上皮屏障的微生物很快被吞噬血细胞吞没和消灭 (关于吞噬细胞生物学的综合回顾见参考14)。当血细胞上的种细胞编码模式识别受体 (Prr) 识别微生物的病原体相关分子模式 (Pampp) 时, 就开始了这一过程。一旦绑定到他们的目标, Prr 启动信号级联, 导致通过肌动蛋白细胞骨架重塑形成假足类。伪足类动物围绕着微生物, 微生物随后被吞没并内化为一个新生的细胞器–噬虫体。当噬虫体被贩运到血细胞内部并通过与溶酶体的一系列相互作用而酸化时, 当噬虫体经历噬虫体成熟的过程时, 微生物就会被破坏。在哺乳动物原代细胞的体外和细胞生物学研究中, 已经有助于识别和表征调节吞噬的因素, 如哺乳动物 fc-γ受体和 c3b 受体15,16。然而, 在哺乳动物系统中, 执行大规模屏幕或体内研究的能力有限。
在这里, 我们提出了一个体内检测在成年果蝇的吞噬, 这是基于一个程序首次推出的大卫·施奈德实验室在 2000年17。施耐德实验室显示, 沿着腹背血管聚集的无柄血细胞很容易产生吞噬聚苯乙烯珠和细菌。为了想象吞噬, 苍蝇被注入荧光标记的颗粒 (如贴有荧光素异硫氰酸酯 (e. colie-fitc) 标签的大肠杆菌), 孵育 30分钟, 让血细胞的时间吞没这些颗粒, 然后再注射色氨酸蓝, 在潜伏期内抑制不吞噬的颗粒的荧光。然后使用倒置荧光显微镜对飞行背侧血管进行成像。这篇开创性的论文, 使用一个相对简单的实验, 证明血细胞吞噬细菌和乳胶珠, 细菌吞噬可以通过预注射苍蝇与乳胶珠, 以及苍蝇没有细胞和体液免疫反应是易受感染的, 甚至大肠杆菌。本报告中介绍的检测方法以施耐德实验室的工作为基础, 通过测量与背血管相关的血细胞所吞没的颗粒的荧光强度来量化体内吞噬作用。
与哺乳动物系统中采用的方法类似,果蝇遗传学家最初使用全基因组的体外 rnai 屏幕来识别细胞免疫反应所需的基因18, 19,20 ,21,22,23。然而, 成人体内吞噬试验的发展使后续实验能够在整个动物中轻松进行, 从而使研究人员能够验证在体外研究中确定的因素的生物学作用。跨膜受体食客的情况就是如此, 它最初被确定为使用 S2 细胞24的 RNAi 筛查中的细菌受体, 后来被证明是介导大肠杆菌(大肠杆菌)、肠球菌粪便和金黄色葡萄球菌(金黄色葡萄球菌) 在成人中的吞噬作用25。
我们的实验室在前瞻性基因屏幕和全基因组关联研究中使用了体内吞噬检测方法 (使用果蝇遗传参考小组 (dgrp)) 来识别调节成人血细胞吞噬功能的新基因。这些研究导致了受体 PGRP-SC1A 和 PGRP-SA26、细胞内囊泡贩运蛋白 rab1427、谷氨酸转运体 polyphemus28和 rna 结合蛋白 fisu-129的表征。
我们预计, 未来的屏幕纳入体内吞噬可能会导致识别额外的基因, 这对果蝇的细胞免疫反应很重要。使用完全测序自交系的屏幕, 如 DGRP 或食子体合成种群资源 (dspr), 可以识别影响吞噬或血细胞发育的自然变异。此外, 这种技术可用于其他果蝇物种, 也可用于筛选新的社区资源, 例如收集国家果蝇物种储存中心 (ndsssc) 维持的 250个果蝇物种) 在康奈尔大学。这些实验可以使用荧光标记的细菌或真菌壁生物制品进行, 这些生物物品可在商业上买到, 也可以使用任意数量的细菌或真菌物种进行–前提是微生物表达荧光标记.
Protocol
Representative Results
Discussion
商业上可用的荧光标记颗粒用于评估一般的吞噬性 (0.2 微米羧改性后期微球) 或微生物的吞噬作用 (荧光标记的热或化学杀生细菌或酵母)。为了评估噬体体组的成熟, 研究人员可以选择带有 ph 敏感染料标记的颗粒, 当 ph 值从中性变低到酸性时, 当噬亚耳溶于酸性时, 这种染料会发光。或者, 为了检查吞噬、病原体识别和吸收的初始步骤, 研究人员应该选择带有荧光标记的粒子, 这些标记在细…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢贝丝·冈萨雷斯博士和阿普拉伊塔·加格博士在进行体内吞噬实验方面提供的支持。NSF UMD 发展种子赠款和 UMD NIH T32 培训赠款、细胞和分子生物学 (CMB) 和宿主病原体相互作用 (HPI) 为这项工作提供了资金。
Materials
| 0.2μm Red Fluorescent Carboxylate Modified FluoSpheres | Invitrogen | F8810 | Fluorescently-labeled latex beads to test general phagocytic capacity of phagocytes. (~580/~605 nm) Inject a 1:20 dilution in PBS with 5% dye. |
| 5430-10 PicoNozzle Kit | World Precision Instruments | 5430-10 | Holder for 1.0mm pipette |
| E. coli (K-12 Strain) BioParticles, Alexa Fluor 488 conjugate | Invitrogen | E13231 | Killed E. coli labeled with Alexa Fluor 488. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~495/~519 nm) |
| E. coli (K-12 Strain) BioParticles, Alexa Fluor 594 conjugate | Invitrogen | E23370 | Killed E. coli labeled with Alexa Fluor 594. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~590/~617 nm) |
| E. coli (K-12 Strain) BioParticles, Fluorescein conjugate | Invitrogen | E2861 | Killed E. coli labeled with FITC (Fluorescein). Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~494/~518 nm) |
| E. coli (K-12 Strain) BioParticles, Texas Red conjugate | Invitrogen | E2863 | Killed E. coli labeled with Texas Red. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~595/~615 nm) |
| E. coli (K-12 Strain) BioParticles, Texas Red conjugate | Invitrogen | E2863 | Killed E. coli labeled with Texas Red. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~595/~615 nm) |
| Needle Pipette Puller | David Kopf Instruments | Model 725 | |
| pHrodo Red E. coli BioParticles Conjugate for Phagocytosis | Invitrogen | P35361 | Killed E. coli labeled with pHrodo Red. Use to test phagocyte reconition, uptake, and phagosome maturation of gram-negative bacteria. (~560/~585 nm). No need to quench with Trypan Blue. |
| pHrodo Red S. aureus BioParticles Conjugate for Phagocytosis | Invitrogen | A10010 | Killed S. aureus labeled with pHrodo Red. Use to test phagocyte reconition, uptake, and phagosome maturation of gram-positve bacteria. (~560/~585 nm). No need to quench with Trypan Blue. |
| Pneumatic PicoPump PV820 | World Precision Instruments | SYS-PV820 | The World Precision Instruments Pneumatic PicoPump PV820 uses differential pressures to hold liquid in the glass needle between injections. The user manually controls short bursts of gas pressure to expel the liquid – allowing delivery of sub-nanoliter volumes. The amount of liquid delivered depends on two main variables – the size of the glass needle opening and the amount of time injection pressure is applied. set the instrument to 100 ms “TIMED” mode. |
| S. aureus (Wood Strain without protein A) BioParticles, Alexa Fluor 488 conjugate | Invitrogen | S23371 | Killed S. aureus labeled with Alexa Fluor 488. Use to test phagocyte recogntion and uptake of gram-positive bacteria. (~495/~519 nm) |
| S. aureus (Wood Strain without protein A) BioParticles, Alexa Fluor 594 conjugate | Invitrogen | S23372 | Killed S. aureus labeled with Alexa Fluor 594. Use to test phagocyte recogntion and uptake of gram-positive bacteria. (~590/~617 nm) |
| S. aureus (Wood Strain without protein A) BioParticles, Fluorescein conjugate | Invitrogen | E2851 | Killed S. aureus labeled with FITC (Fluorescein). Use to test phagocyte recogntion and uptake of gram-positive bacteria. (~494/~518 nm) |
| Thin Wall Glass Capillaries | World Precision Instruments | TW100F-3 | Needles for injection. OD = 1.0 mm |
| Trypan Blue Solution (0.4%) | Sigma | T8154 | Used to quench extracellular fluorescence of Fluorescein, Alexa Fluor, or Texas Red labeled particles. |
| ZEISS SteREO Microscope (Discovery.V8) | Zeiss | SteREO Discovery.V8 | Inverted fluorescence microscope for imaging flies. Use a digital camera (example: AxioCam HC camera) and the accompanying software (example: AxioVision 4.7 software) to take pictures. |
| Zymosan A (Saccharomyces cerevisiae) BioParticles, Alexa Fluor 488 conjugate | Invitrogen | Z23373 | Killed labeled with Alexa Fluor 488. Use to test phagocyte recogntion and uptake of yeast. (~495/~519 nm) |
| Zymosan A (Saccharomyces cerevisiae) BioParticles, Alexa Fluor 594 conjugate | Invitrogen | Z23374 | Killed labeled with Alexa Fluor 594. Use to test phagocyte recogntion and uptake of yeast. (~590/~617 nm) |
| Zymosan A (Saccharomyces cerevisiae) BioParticles, Fluorescein conjugate | Invitrogen | Z2841 | Killed labeled with FITC (Fluorescein). Use to test phagocyte recogntion and uptake of yeast. (~494/~518 nm) |
| Zymosan A (Saccharomyces cerevisiae) BioParticles, Texas Red | Invitrogen | Z2843 | Killed labeled with Texas Red. Use to test phagocyte recogntion and uptake of yeast. (~595/~615 nm) |
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