Summary

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

Published: November 27, 2014
doi:

Summary

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Abstract

对粘膜病原体的早期防御由双方的上皮屏障和先天免疫细胞。两者的免疫竞争,他们的互通,是极为重要的保护,防止感染。上皮细胞和先天免疫细胞与病原体的相互作用被最佳研究在体内 ,其中复杂的行为展开在时间和空间。然而,现有的模式不允许与病原体战斗在粘膜水平易时空成像。

这里开发的模型创建粘膜感染通过直接注射的真菌病原体, 白色念珠菌 ,进入斑马鱼幼鱼的鳔的。所得感染使上皮和先天免疫细胞行为高分辨率成像贯穿粘膜病的发展。该方法的多功能性允许主机的询问来探测免疫事件导致的pH的详细序列agocyte招募和检查特定的细胞类型并在保护分子途径中的作用。此外,病原菌的免疫攻击的功能的行为可以同时使用荧光蛋白表达C.成像白色念珠菌 。所述宿主 – 病原体相互作用的增加的空间分辨率,也可以使用所描述的快速鳔夹层技术。

这里所描述的粘膜感染模型很简单,重现性好,使其成为粘膜念珠菌病的研究中一个有价值的工具。该系统还可以是大致平移的其他粘膜病原体如结核分枝杆菌,细菌或病毒的微生物,通常通过上皮表面感染。

Introduction

Mucosal infections can lead to life threatening bloodstream infections due to the damage of the epithelial barrier, which allows pathogens access to the systemic environment1,2. In addition, mucosal infections can also cause significant immunopathology even when contained externally3-5. The commensal unicellular fungus Candida albicans is present in the majority of the population in the oral cavity and other mucosal sites6-9. Although normally contained by innate and adaptive immune responses, innate immune defects and medical interventions can lead to severe mucosal candidiasis. The assault on the epithelial barrier results in an increased risk of life threatening disseminated disease as well as immunopathology, as in the case of vulvo-vaginal candidiasis, additionally C. albicans colonization has been linked with lung immune homeostasis10,11. Disseminated candidiasis is now the fourth most common bloodstream infection in intensive care units12 and mortality as high as 40% makes it a major concern. Due to the increase in immunomodulatory treatments for patients with autoimmune diseases, cancer or organ transplants, it is imperative to understand the interaction between this pathogen and the mucosal immune compartment.

The majority of cell biological advances regarding C. albicans-cell interactions at the mucosal level come from in vitro13-15 and murine models16-18. Both these approaches have distinct advantages, but the ability to image live cells at high resolution in an intact host has limited the temporal and spatial characterization of the infection. For these studies, there is the need for an in vivo model where the interaction of pathogen, innate immune and epithelial cells can be visualized in an intact vertebrate host.

The zebrafish has emerged as an invaluable tool for the understanding of human disease, mainly due to its transparency and amenability to genetic manipulation. Cell and organ development have been imaged in exquisite detail, which has led to the description of novel immune cell behaviors, such as T cell behavior in the developing thymus19 or the battle between intracellular mycobacteria and phagocytes20-22. Recent work has described intestinal microbe-host interactions in zebrafish and shown that microbial colonization of the intestinal tract affects host intestinal physiology and resistance to other infections23,24. Furthermore, infection through the gut epithelium has been described for several pathogens.

In contrast to the intestinal tract, the swimbladder represents a more isolated and complementary mucosal model. This organ is an extension of the developing gut tube and forms anteriorly to the liver and pancreas25,26. It produces surfactant, mucus and antimicrobial peptides27,28 and anatomically, as well as ontogenetically, this organ is considered a homologue of the mammalian lung29,30. Since the pneumatic duct remains connected to the gut in the zebrafish, this allows for immersion infection to occur naturally. Remarkably, the only known naturally occurring infections of fish with Candida species are C. albicans infections in the swimbladder31. We recently described an experimental immersion infection model where C. albicans infects the swimbladder, and found that this infection recapitulates some of the hallmarks of C. albicans-epithelial interaction in vitro32,33.

In the method presented here, the original immersion infection model is improved by directly injecting C. albicans into the swimbladder of 4 days post fertilization (dpf) zebrafish. This allows for precise temporal control of infection as well as a highly reproducible inoculum. It permits detailed intravital imaging, coupled with the versatility of the zebrafish model. As an example of what can be done with this method, we present the spatio-temporal dynamics of C. albicans growth along with neutrophil recruitment to the site of infection. Because zebrafish swimbladder tissue is challenging to image intravitally, we also present a rapid swimbladder dissection technique that improves fluorescence signal and microscopic resolution. These methods expand the toolbox for fungal, immunological, and aquaculture research as well as describing a novel infection route that may be translated to model other fungal, bacterial or viral infections of mucosal surfaces.

Protocol

注:所有斑马鱼护理协议和实验按照根据机构动物护理和使用委员会(IACUC)协议A2012-11-03 NIH的指导方针进行。 1.斑马鱼饲养到4天受精后收集AB斑马鱼,或任何其他的转基因系中,第一3小时受精后内,如图另一视频34。 120孵育鸡蛋含有150ml E3媒体15cm的培养皿(5毫米氯化钠,0.17毫米氯化钾,0.33毫米氯化钙2; 0.33毫米氯化镁2 2毫米的HEPES,…

Representative Results

在注射后鳔这里介绍的实验方法描述在注射C的一致剂量白念珠菌的酵母细胞在4鳔DPF斑马鱼。以前的工作与浸入式模型表明,到C的鳔免疫反应白色念珠菌是类似于哺乳动物粘膜念珠菌32。在这里,我们展示了修改后的感染的方法是比较简单的,重复性好,快速;几百斑马鱼的可以注射和内几个小时进行筛选。 所述鳔的准确?…

Discussion

在鳔显微注射疾病模型的研究进展和限制

这里提出的模型是在Gratacap 等人描述的粘膜念珠菌浸没模型的扩展(2013)。它增加了一个控制感染时间的优点,一个高度重复性感染的剂量,并因此提高效率。我们在这里表明,允许感染动力学的非常详细的非侵入性颞文档以及鳔的更高分辨率离体成像的新方法。这些程序应有利于C的研究白色念珠菌 -in…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

作者感谢乐郑氏博士和博士托宾慷慨地提供了α-catenin的:黄水晶鱼线和比尔·杰克曼允许我们做的拍摄在他的实验室。作者承认的资金来源国立卫生研究院(资助5P20RR016463,8P20GM103423和R15AI094406)和美国农业部(项目#ME0-H-1-00517-13)。此稿件发布为主要农林试验站出版物号3371。

Materials

Name Company Catalog Number Comments
1.7 mL tubes Axygen MCT-175-C
Deep Petri dishes Fisher Scientific 89107-632
Transfer pipettes Fisher Scientific 13-711-7M
Yeast Extract VWR Scientific 90000-726
Peptone VWR Scientific 90000-264
Dextrose Fisher Scientific D16-1
Agar VWR Scientific 90000-760
Fine tweezers (Dumont Dumoxel #5) Fine Science Tools 11251-30
Wooden Dowels VWR Scientific 10805-018
Low Melt Agarose VWR Scientific 12001-722
Flaming Brown Micropipette Puller Sutter Instruments P-97
Borosilicate capillary Sutter Instruments BF120-69-10
MPPI-3 Injection system Applied Scientific Instrumentation MPPI-3
Back Pressure Unit Applied Scientific Instrumentation BPU
Micropipette Holder kit Applied Scientific Instrumentation MPIP
Foot Switch Applied Scientific Instrumentation FSW
Micromanipulator Applied Scientific Instrumentation MM33
Magnetic Base Applied Scientific Instrumentation Magnetic Base
Tricaine methane sulfonate Western Chemical Inc. MS-222
Dissecting Scope Olympus SZ61 top SZX-ILLB2-100 base
Confocal Microscope Olympus IX-81 with FV-1000 laser scanning confocal system
20x microscope objective Olympus UPlanSApo 20x/0.75
Roller drum New Brunswick Scientific TC-7
Microloader pipette tips Eppendorf 930001007
Glass culture tubes (16 x 150 mm) VWR Scientific 60825-435
NaCl VWR Scientific BDH4534-500GP
KCl VWR Scientific BDH4532-500GP
MgSO4 VWR Scientific BDH0246-500GP
HEPES (Corning) VWR Scientific BDH4520-500GP
Children clay (Play-Doh) Hasbro
CaCl2 Fisher Scientific C69-500
Methylene Blue VWR Scientific VW6276-0
PTU Sigma P7629-10G
Petri dishes Fisher Scientific FB0875712
Hemocytometer (Hausser scientific) VWR Scientific 15170-172
Type A immersion oil Blue Marble Products 51935
Centrifuge Eppendorf 5424
Vortex Genie VWR Scientific 14216-184
Agarose (Lonza) VWR Scientific 12001-870
Na2HPO4 Fisher Scientific S374-500
KH2PO4 Fisher Scientific P285-500
Fishing wire Stren
96 well imaging plate (Sensoplate) Greiner Bio-One 655892
High vacuum grease (Dow Corning) VWR Scientific 59344-055
Microslide (25 x 75 mm) VWR Scientific 48300-025
Cover slips (18 x 18 mm), No 1.5 VWR Scientific 48366-045
15 cm Petri dish (Olympus plastics) Genesee Scientific 32-106
Glycerol (EMD chemicals) VWR Scientific EMGX0185-5
24-well culture dish (Olympus plastics) Genesee Scientific 25-107
Weight boats (8.9 cm) VWR Scientific 89106-766

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Gratacap, R. L., Bergeron, A. C., Wheeler, R. T. Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection. J. Vis. Exp. (93), e52182, doi:10.3791/52182 (2014).

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