Modellierung Mucosal Candidiasis in Zebrafisch-Larven von Schwimmblase Injection
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
Frühe Verteidigung gegen Krankheitserreger Schleimhaut besteht sowohl eine Epithelbarriere und angeborenen Immunzellen. Die immunocompetency von beiden, und ihre Zwischenverbindung, an erster Stelle stehen für den Schutz vor Infektionen. Die Wechselwirkungen von Epithelzellen und Zellen des angeborenen Immunsystems mit einem Pathogen besten in vivo, wo komplexe Verhalten entfaltet über Zeit und Raum untersucht. Allerdings sind bestehende Modelle nicht für den einfachen Raum-Zeit-Bildgebung der Kampf mit Krankheitserregern an der Schleimhautniveau ermöglichen.
Die hier entwickelten Modell erstellt eine Schleimhaut-Infektion durch direkte Injektion des Pilzpathogen, Candida albicans, in die Schwimmblase der jugendlichen Zebrafisch. Die daraus resultierende Infektion ermöglicht hochauflösende Bildgebung von epithelialen und angeborenen Immunzellverhaltens in der gesamten Entwicklung der Mucosal Disease. Die Vielseitigkeit dieser Methode ermöglicht die Abfrage des Host, um die detaillierte Abfolge der Ereignisse, die zu Immun pH-Sondeagocyte Rekrutierung und die Rollen von bestimmten Zelltypen und molekulare Signalwege in Schutz zu untersuchen. Darüber hinaus kann das Verhalten des Erregers als Funktion der Immunangriff gleichzeitig unter Verwendung fluoreszierende Protein exprimierenden C. abzubildenden albicans. Erhöhte räumliche Auflösung der Wirt-Pathogen-Interaktion ist auch möglich, mit dem beschriebenen schnellen Schwimmblase Dissektionstechnik.
Die hier beschriebene Schleimhaut-Infektionsmodell ist einfach und sehr gut reproduzierbar, so dass es ein wertvolles Werkzeug für die Untersuchung der Schleimhautinfektionen. Dieses System kann auch allgemein übersetzbar andere Schleimhautpathogene, wie beispielsweise mykobakteriellen, bakterielle oder virale Keime, die normalerweise durch epitheliale Oberflächen infizieren.
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
Representative Results
Discussion
Fortschritte und Grenzen der Schwimmblase Mikroinjektion Krankheitsmodell
Das hier vorgestellte Modell ist eine Erweiterung des in Gratacap et al Schleimhaut Candidiasis Tauch Modell (2013). es fügt die Vorteile eines kontrollierten Infektion Zeit eine hoch reproduzierbare Infektionsdosis und damit eine verbesserte Effizienz. Wir zeigen hier neue Methoden, die nicht-invasive zeitliche Dokumentation der Infektionsdynamik im Detail als auch eine höhere Auflösung Ex-vivo-B…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Die Autoren danken Dr. Le Trinh und Dr. Tobin für die großzügige Bereitstellung der α-Catenin: Citrin Angelschnur und Bill Jackman für die Erlaubnis, die Dreharbeiten in seinem Labor zu tun. Die Autoren erkennen die Finanzierungsquellen National Institutes of Health (Grants 5P20RR016463, 8P20GM103423 und R15AI094406) und USDA (Projekt # ME0-H-1-00517-13). Dieses Manuskript ist als Haupt Land- und Forstwirtschaft Experiment Station Veröffentlichungsnummer 3371 veröffentlicht.
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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