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.
Tidlig forsvar mod mucosale patogener består af både en epitelbarriere og medfødte immunceller. Den immunocompetency af begge, og deres indbyrdes kommunikation, er altafgørende for at beskytte mod infektioner. Interaktionerne af epitel- og medfødte immunceller med et patogen bedst undersøgt in vivo, hvor kompleks adfærd udfolder i tid og rum. Men de eksisterende modeller ikke mulighed for nem spatio-temporale billeddannelse af kampen med patogener på slimhinde-niveau.
Den udviklede model her skaber en mucosal infektion ved direkte injektion af det fungale patogen, Candida albicans, i swimbladder af unge zebrafisk. Den resulterende infektion giver høj opløsning billeddannelse af epitel- og medfødte immunsystem celle adfærd i hele udviklingen af mucosal disease. Alsidigheden i denne metode giver mulighed for afhøring af værten til at undersøge detaljeret sekvens af immune begivenheder, der fører til pHagocyte rekruttering og undersøge roller bestemte celletyper og molekylære veje i beskyttelse. Desuden kan adfærd patogen som en funktion af immunangreb skal afbildes samtidigt ved hjælp af fluorescerende protein-udtrykkende C. albicans. Øget rumlige opløsning vært-patogen interaktion er også muligt ved hjælp af den beskrevne hurtige swimbladder dissektion teknik.
Det mucosal infektion model her beskrevne er ligetil og meget reproducerbar, hvilket gør det et værdifuldt redskab for studiet af mukøs candidiasis. Dette system kan også være stort set translaterbare andre mucosale patogener, såsom mycobakterielle, bakterielle eller virale mikrober, der normalt inficerer gennem epiteloverflader.
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.
Forskud og begrænsninger i swimbladder mikroinjektion sygdomsmodel
Modellen præsenteres her er en forlængelse af slimhinde-candidiasis fordybelse model beskrevet i Gratacap et al (2013).; det tilføjer fordelene ved en kontrolleret infektion tid, en meget reproducerbar infektion dosis og derfor forbedret effektivitet. Vi viser her nye metoder, der tillader ikke-invasiv tidsmæssig dokumentation af infektion dynamik i stor detalje samt højere opløsning ex vivo billeddannel…
The authors have nothing to disclose.
Forfatterne takker Dr. Le Trinh og Dr. Tobin for generøst giver den α-catenin: citrin fisk linje og Bill Jackman for at tillade os at gøre optagelserne i sit laboratorium. Forfatterne anerkender finansieringskilderne National Institutes of Health (Grants 5P20RR016463, 8P20GM103423 og R15AI094406) og USDA (Projekt # ME0-H-1-00517-13). Dette håndskrift er offentliggjort som Main landbrug og skovbrug Experiment Station publikation nummer 3371.
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 |