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 h....

Protocol

This protocol has been approved by the Mississippi State University Institutional Animal Care and Use Committee (MSU-IACUC). All zebrafish used in this study were bred from a homozygous colony of rag1^-/- mutant zebrafish previously established in the specific pathogen-free hatchery in the College of Veterinary Medicine, Mississippi State University (MSU)¹⁰. Wild-type zebrafish were also propagated in this hatchery. In these studies, toxin exposures can be performed in vivo<.......

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

Materials

Name	Company	Catalog Number	Comments
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