Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

Summary

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

Abstract

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

Introduction

The immune system is comprised of two branches: innate and adaptive immunity. Innate immunity is evolutionarily more ancient than adaptive immunity. Invertebrates are presently thought to have only innate immunity, whereas vertebrates possess both the innate and adaptive branches. While adaptive immunity confers specific and long-lasting immunity to certain pathogens, innate immunity is an immediate response to invading bacteria, viruses, and fungi. A crucial aspect of the innate immune response involves the release of cytokines and chemokines, which results in inflammation and recruitment of phagocytes (e.g. macrophages, neutrophils) to engulf and destroy fo....

Protocol

1. Zebrafish Care and Maintenance

1. Husbandry: Mass spawn adult zebrafish as previously described¹³. Collect spawned embryos as previously described¹⁴.
2. Microinjection (if desired): Microinject 1-4 cell stage zebrafish embryos with morpholino oligonucleotides to knockdown gene products or mRNA to overexpress gene products as previously described¹⁵.
   1. Maintain an adequate pool of mock injected controls (at least 48 living, mock injected control fish and 48 living, experimentally manipulated fish are needed to fill a 96 well microplate).
3. Maintain embryos: Grow embryos in deep petri dis....

Results

Here, we provide data comparing the respiratory burst response in zebrafish embryos (wild-type, AB background) at 48 and 72 hours post fertilization (hpf). The 48 hpf embryos acted as our control group and the 72 hpf embryos as our experimental group. The sample size used was 24 un-induced embryos and 24 PMA-induced embryos per developmental stage. Raw fluorescence readings (in Relative Fluorescence Units (RFU)) were obtained by reading the microplate 4 hours after the addition of PMA. Raw fluorescence values are provide.......

Discussion

The primary function of phagocytes is to detect, engulf, and destroy pathogens. The ability of phagocytes to produce an adequate respiratory burst is critical for this function. Thus, quantification of the respiratory burst response is one method to allow comparison of general innate immune health and function between groups of individuals and/or in response to experimental manipulations. Here, we describe a protocol to induce, quantify, and compare the respiratory burst response between groups of individual zebraf.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Instant Ocean Sea Salt	Instant Ocean	SS15-10
H2DCFDA	Sigma Aldrich	35845-1G
PMA	Fisher	BP6851
DMSO	Sigma Aldrich	D2438-5X10ML
Tricaine S MS222	Western Chemical	100 grams
DMEM/F-12, No Phenol Red	Life Technologies	11039-021
Deep Petri Dishes	VWR	89107-632
Plastic Transfer Pipettes	Fisher	13-711-7M
#5 Dumont Forceps	Electron Microscopy Sciences	72700-D
1.7 ml Micro Centrifuge Tubes	Axygen	10011-724
15 ml Conical Centrifuge Tubes	VWR	21008-918
5 ml Serological Pipettes	Greiner Bio One	606180
Synergy 2 Multi-Mode Microplate Reader	BioTek	Contact BioTek
Black 96 Well Microplate	VWR	82050-728
25 ml Sterile Reservoirs	VistaLab	3054-2003
P200 Pipettor	Gilson	F123601
Multichannel Pipettor	VWR	89079-948
Pipette Tips	VWR	89079-478