Evaluating the Effects of Biotoxins on Immune Cell Functions in Zebrafish
Summary
This protocol describes flow cytometric assays that can evaluate the effects of toxin exposure on the endocytic functions of different subpopulations of zebrafish leukocytes. The use of specific functional inhibitors in the assay allows differentiation of the altered endocytic mechanisms.
Abstract
A variety of biological toxins can be present at harmful levels in the aquatic environment. Cyanobacteria are a diverse group of prokaryotic microorganisms that produce cyanotoxins in the aquatic environment. These biotoxins can be hepatotoxins, dermatoxins, or neurotoxins and can affect fish and mammals. At high levels, these compounds are fatal. At non-lethal levels, they act insidiously and affect immune cell functions. Algae-produced biotoxins include microcystin and anatoxin A. Aquatic animals can also ingest material contaminated with botulinum neurotoxin E (BoNT/E) produced by Clostridium botulinum, also resulting in death or decreased immune functions. Zebrafish can be used to study how toxins affect immune cell functions. In these studies, toxin exposures can be performed in vivo or in vitro. In vivo studies expose the zebrafish to the toxin, and then the cells are isolated. This method demonstrates how the tissue environment can influence leukocyte function. The in vitro studies isolate the cells first, and then expose them to the toxin in culture wells. The leukocytes are obtained by kidney marrow extraction, followed by density gradient centrifugation. How leukocytes internalize pathogens is determined by endocytic mechanisms. Flow cytometry phagocytosis assays demonstrate if endocytic mechanisms have been altered by toxin exposure. Studies using isolated leukocytes to determine how toxins cause immune dysfunction are lacking. The procedures described in this article will enable laboratories to use zebrafish to study the mechanisms that are impacted when an environmental toxin decreases endocytic functions of immune cells.
Introduction
There are many types of environmental biotoxins and immune suppressive agents. Algae blooms that contain bacterial toxins occur in inland waters and can also occur as biofilms1. Cyanobacteria (blue-green algae) naturally occurs in all freshwater ecosystems. Cyanobacterial blooms have substantially increased in freshwater systems2. At certain times, the Cyanobacteria can produce toxins that are harmful to aquatic and terrestrial animals. These toxins can affect the liver, skin, and mucous membranes, and/or the nervous system. Two compounds produced by Cyanobacteria are microcystin and anatoxin A. Microcystin is a cyclic heptapeptide3. Anatoxin A is an alkaloid4. Botulinum neurotoxin E (BoNT/E) is another toxin that occurs in aquatic systems. It is produced by Clostridium botulinum and can be ingested by aquatic animals5.
Exposure to environmental toxins affects fish and can also affect animal health and increase disease occurrences6. Understanding how these toxins affect immune cells is fundamental to determination of the risks associated with exposure to these substances. Zebrafish are an excellent model for studying the effects of environmental toxins on immune cells7. Developing a method that utilizes flow cytometry and zebrafish leukocytes is highly beneficial. Zebrafish have physiological relevance to humans, and this method can be applied to a wide range of research areas, from basic toxicology and immunology to drug discovery and developmental biology. Because they are aquatic organisms, zebrafish are particularly suitable for studying the effects of waterborne environmental toxins7. The use of zebrafish is less expensive than other vertebrate models, and their use raises fewer ethical concerns.
White blood cells, or leukocytes, are the first line of cellular defense against disease causing organisms. Endocytosis is the process of a cell taking up or internalizing a liquid or particle that is external to the cell. This is accomplished by the cell enclosing the compound in a vesicle8. Leukocytes use this process as the first step in killing pathogens and preparing a defense against disease. Phagocytosis is a type of endocytosis and was one of the first methods used to investigate the effects of environmental pollutants on fish health9. The Petrie-Hanson lab has developed methods using zebrafish leukocytes to screen biotoxins for their potential ability to interfere with leukocyte endocytic and phagocytic functions and impact immune defenses. The types of endocytosis included in these methods are pinocytosis, phagocytosis, calcium dependent receptor-mediated phagocytosis and mannose receptor mediated phagocytosis. Using flow cytometry methods with zebrafish were first described in the Petrie-Hanson lab9 and are used routinely to investigate aquatic toxins and pathogens. Rag1-/- mutant zebrafish do not have T and B cells10 and can be used to specifically investigate innate immune cell mechanisms.
Flow cytometry is laser-based and can be used to determine the physical properties of cells. The forward scatter, or FSC value is plotted on the X axis and represents the size of the cell. The side scatter, or SSC, is plotted on the Y axis and represents the cytoplasmic granularity of the cell. The resulting plot demonstrates populations of cells with similar physical characteristics grouped together, with the different cell types appearing at various locations on a scatter plot. These populations can change location on the scatter plot as the physical characteristics of the cells change9. Using this technique with zebrafish leukocytes enables researchers to assess changes in cell populations in response to various stimuli, including environmental toxins.
The flow cytometer is multi-dimensional, and multiple types of fluorophores can be used in the evaluation to further characterize the cells and their activity. In the assays described in this protocol, endocytosis is characterized by measuring the amount of fluorescent material a cell has internalized. If and how toxin exposure affects endocytic mechanisms can be determined by comparing the ability of toxin exposed cells to take up the material compared to the ability of non-toxin exposed cells using flow cytometry. The endocytic processes that can be evaluated this way include pinocytosis, receptor mediated endocytosis and phagocytosis.
Pinocytosis is the uptake of soluble components, and it does not utilize cell receptors. Uptake involves cytoplasmic rearrangement by microfilaments and microtubules to form small vacuoles. Luciferase Yellow (LY) is a fluorescent dye used to measure liquid uptake by non-selective pinocytosis11. Receptor mediated endocytosis involves the selective uptake of large molecules. Fluorescein (FITC) labeled dextran (DX) 40 can be used to evaluate this process. Phagocytosis is a form of endocytosis that ingests particles greater than 0.5 micrometers. This process is investigated by procedures using FITC-DX70 and FITC-Eschericia coli. DX40 and DX70 have molecular weights of 40,000 and 70,000, respectively. FITC-E. coli is the standard laboratory strain of E. coli bound to a fluor that can be measured by the flow cytometer. Many forms of the receptor mediated endocytosis require calcium as a signaling molecule and for cytoskeletal rearrangement9. Another type of receptor mediated endocytosis is mannose receptor (MR) mediated endocytosis. Mannose receptors are transmembrane proteins that recognize forms of mannan on microbial cell surfaces9. To optimize these procedures, a dose response curve should be created with each toxin to establish the doses to be used. A saturation curve should be performed for LY, FITC-DX40, FITC-DX70 and FITC-E. coli to assess the correct concentration to use.
The mechanisms used by leukocytes to internalize different particles may vary. To suggest which component of the process may be affected by toxin exposure, inhibitors can be added to block the phagocytic mechanisms. Cytochalasin D (CCD) will inhibit microtubule movement and therefore, pinocytosis. CCD does not influence receptor-mediated endocytosis11. EDTA blocks calcium (Ca2+) dependent receptor-mediated endocytosis. Mannan is a natural ligand for the MR. Mannan is used as a mannose receptor inhibitor to assess if phagocytosis or pinocytosis is mannose receptor mediated9.
The purpose of this protocol is to demonstrate the procedures for determining whether toxin exposure has affected the ability of phagocytic leukocytes to uptake pathogens. These protocols may also discern if a specific endocytic mechanism is affected. Performing these assays on the flow cytometer allows further discrimination by selecting leukocyte populations based on size and cytoplasmic granularity to determine if leukocyte subpopulations have been differentially affected. This method relies on electronic gating of cell populations.
Protocol
Representative Results
Discussion
Utilizing flow cytometry with zebrafish leukocytes offers a powerful and versatile approach for studying the immune system in detail, assessing the impact of environmental toxins, and facilitating toxicological research. It provides a way to quickly and effectively evaluate the impact of toxins on immune cells and the immune response. The results reveal humoral factors involved and suggests how the fish’s physiology and metabolism interact with environmental biotoxins. The overview of the protocol is depicted in <strong …
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors thank Izak Hanson for the daily maintenance of the zebrafish used, and Treva Billyard and Sterling Bailey for assistance in proofing and formatting this manuscript.
Materials
| 10% fetal bovine serum | Gibco | A3160501 | |
| 14 mL round bottom centrifuge tubes | BD Biosciences | 352059 | |
| 40 µm cell straininer | Corning | 07-201-430 | |
| 5 mL flow cytometry tubes | BD Biosciences | 352235 | |
| 50 mL conical centrifuge tube | Corning | 14-959-49A | |
| Absolute ethanol | Fisher | BP2818-500 | |
| Automated cell counter | Life Technologies Countess II FL | for studying cell viability | |
| Bovine serum albumin (BSA) | Sigma | A3059 | |
| cytochalasin D (CCD) | Sigma | C8273-5MG | |
| Dextran 40 | Sigma | FD40-100MG | |
| Dextran 70 | Sigma | 46945-100MG-F | |
| Escherichia coli DH5α (or other lab bacterial strain) | New England Biolabs | C29871 | |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma | ED4SS | |
| Flow analysis software | Novoacea software | ||
| Flow cytometer | Novocyte 3000 | ||
| Fluorescein | Fluka BioChemica | 46950 | |
| hanks balanced salt solution without calcium or magnesium | Sigma | H4891 | |
| Histopaque 1077 | Sigma | 10771-100ML | |
| Lucifer Yellow | Sigma | L0259-25MG | |
| Mannan | Sigma | M7504-250MG | |
| Phosphate buffered saline | Sigma | P3813 | |
| RPMI-1640 with GlutaMax | Gibco | 61870036 | |
| Statistical software | SPSS | ||
| Toxin | |||
| Tricaine | Western Chemical Inc | NC0342409 |
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