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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 foreign invaders.

Successful innate immune responses involve: (1) recognition of invading microorganisms; (2) induction of the appropriate signaling cascades (e.g. release of cytokines and chemokines); (3) proper development/adequate numbers of phagocytic cells; (4) migration of phagocytes to sites of infection; (5) engulfment of pathogens; and (6) destruction of engulfed microorganisms. A deficiency in any one of these steps could lead to the host being overwhelmed by, and succumbing to, the infection. A robust innate immune response is vital to the health of organisms because it is the first line of defense against pathogens in all plants and animals. In vertebrates, it also potentiates the adaptive immune response¹. Therefore, it is critical that we are able to evaluate all aspects of the innate immune response in order to better understand it and to optimize its function.

Many model organisms are used to study innate immunity, ranging from Arabadopsis to C. elegans to Drosophila to mice to cultured human cells. An advantage to using the zebrafish (Danio rerio) model system to study innate immunity is that the zebrafish is a vertebrate, with both innate and adaptive immunity, yet the development of innate and adaptive immunity are temporally segregated. Zebrafish rely solely on innate immunity for protection against infection until adaptive immunity becomes fully functional, which occurs around 4-6 weeks post fertilization². In addition to tools for genetic manipulation, optical clarity and rapid, external development, innate immunity as the principle mode of defense in zebrafish embryos provides a simplified model in which to study the complexities of the innate immune response in vivo.

Multiple protocols have been developed to assess different facets of the innate immune response in zebrafish embryos. Microarrays and RNAseq have validated that the cytokine profiles elicited by the zebrafish innate immune response are similar to that of humans and have also suggested the involvement of unexpected genes in innate immunity^3,4. The transparency of the zebrafish embryo and fluorescent, transgenic strains of pathogens and zebrafish allow for visualization of dynamic host-pathogen interactions in vivo in real time. Transgenic zebrafish embryos expressing GFP under control of the neutrophil-specific myeloperoxidase promoter^5,6 or the macrophage-specific mpeg1 promoter⁷ have made it possible to visualize and quantify phagocyte migration to sites of localized infections⁸ as well as to visualize phagocytosis and destruction of fluorescently labeled pathogens^8,9. Zebrafish embryos are also amenable to the generation of high-throughput assays and chemical screens. Accordingly, high-throughput methods of transcriptome analysis upon infection¹⁰ and phagocyte migration to sites of chemically induced injury¹¹ have recently been developed.

Of the techniques listed above, none quantitatively assess the final stage of pathogen destruction by phagocytes. This final stage involves a respiratory burst (i.e. production of ROS and other toxic compounds), which kill the engulfed pathogens. The enzyme NADPH oxidase is a major source of ROS in phagocytic cells. Assembly of the subunits of the NADPH oxidase enzyme results in transfer of electrons to oxygen, generating superoxide anions. Through subsequent enzymatic reactions, superoxide can then be converted into hydrogen peroxide and hypochlorous acid (Figure 1A). It is the respiratory burst of phagocytes that kills pathogens and thus, the quantification of the respiratory burst potential of zebrafish embryos is indicative of overall innate immune health. We developed a fluorescence-based assay to quantify the respiratory burst in groups of individual zebrafish embryos¹². This assay utilizes the non-fluorescent, reduced form of a commercially available, cell-permeable dye. This dye, 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), is converted into the fluorescent compound, 2',7'-dichlorofluorescein (DCF), upon oxidation. The diverse ROS generated by the phagocyte respiratory burst can oxidize H2DCFDA and generate fluorescence²⁴. The appearance of fluorescence can be used to quantify and compare the respiratory burst response between groups of zebrafish. The protein kinase C agonist phorbol myristate acetate (PMA) is used to chemically induce NADPH oxidase to produce ROS and thus increase fluorescence readings (Figure 1B). Herein, we provide a detailed protocol of a modified and optimized version of this zebrafish embryo respiratory burst assay. This assay can be used to compare the respiratory burst between groups of individual zebrafish embryos over time and/or in response to experimental manipulations (e.g. morpholino-mediated protein knockdown). The use of this method, in conjunction with other zebrafish innate immunity assays, will provide a more complete picture of the complex and critical innate immune response.

Protokół

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 dishes at 28 °C in egg water (60 μg/ml Instant Ocean Sea Salt in distilled water-autoclaved) until the desired developmental stage (a respiratory burst response is not detectable using this protocol in zebrafish embryos younger than 2 days post fertilization¹²). Note: It has been observed that preventing pigmentation in zebrafish embryos through either the use of 1-phenyl 2-thiourea (PTU) or golden/slc24a5 mutant zebrafish does not significantly alter the induction of fluorescence by PMA (unpublished data, embryos tested at 48, 72, and 96 hpf).
   1. Remove dead embryos daily with a plastic transfer pipette.
   2. Carefully decant old egg water and replenish with new egg water daily.
4. Dechorionate embryos: On the day of the experiment, dechorionate embryos (if embryos are still in their chorions) as previously described using two fine forceps¹⁴ (this respiratory burst assay can also be performed on dissected kidneys from adult zebrafish¹² and detailed protocols for kidney dissection from adult zebrafish have been previously described^16,17).

2. Solution Preparation

1. Prepare a stock solution of H2DCFDA: Weigh out 1 mg of H2DCFDA.
   1. Dissolve 1 mg of H2DCFDA in 1 ml of dimethyl sulfoxide (DMSO) to make a 1 mg/ml stock solution.
   2. Make 22 μl aliquots of this stock solution in 1.7 ml microcentrifuge tubes.
   3. Wrap the aliquots of H2DCFDA stock solution in aluminum foil and keep in the dark whenever possible because H2DCFDA is light sensitive.
   4. Store H2DCFDA aliquots at -20 °C for up to 3 months.
2. CAUTION - Prepare a stock solution of phorbol myristate acetate (PMA): Weigh out 1 mg of PMA using proper personal protective equipment (i.e. gloves, goggles and mask).
   1. Dissolve PMA in 1 ml of DMSO to make a 1 mg/ml stock solution.
   2. Make 11 μl aliquots in 1.7 ml microcentrifuge tubes.
   3. Store aliquots of PMA stock solution at -80 °C for up to 3 months.
3. Prepare a working solution of H2DCFDA: On the day of the experiment, make a H2DCFDA working solution by adding 1 part H2DCFDA stock solution to 1 part DMSO (500 μg/ml H2DCFDA final concentration). For example, add 20 μl of H2DCFDA stock solution and 20 μl DMSO to a foil wrapped 1.7 ml microcentrifuge tube.
4. Prepare a working solution of PMA: On the day of the experiment, make a PMA working solution by adding 1 part PMA stock solution to 49 parts nuclease free water (20 μg/ml PMA final concentration). For example, dilute 10 μl PMA stock solution in 490 μl nuclease free water in a 1.7 ml microcentrifuge tube.
5. Prepare a dosing solution of H2DCFDA: On the day of the experiment, make a H2DCFDA dosing solution with a final concentration of 1 μg/ml H2DCFDA in egg water. The prepared volumes of the dosing solutions can be modified as desired, but ensure that the final concentrations of the reagents are maintained. For kidneys, instead of egg water, use the same volume of Dulbecco's modified Eagle's medium/F-12 (50% DMEM, 50% F-12, without phenol red). To make 5 ml of H2DCFDA dosing solution, use a 5 ml serological pipette to transfer 5 ml of egg water (or DMEM/F-12) into a 15 ml conical centrifuge tube wrapped in foil and labeled 'H'.
   1. Remove 10 μl of egg water (or DMEM/F-12) from the 15 ml conical tube labeled 'H' and discard.
   2. Add 10 μl of H2DCFDA working solution into the 15 ml conical tube labeled 'H' and vortex to mix (this volume is enough for 48 embryo or kidney samples (half of a full 96 well microplate) and these wells will provide measurements of the level of background fluorescence).
6. Prepare a dosing solution of H2DCFDA + PMA: On the day of the experiment, make a H2DCFDA + PMA dosing solution with final concentrations of: H2DCFDA - 1 μg/ml and PMA - 400 ng/ml. To make 5 ml of H2DCFDA + PMA dosing solution, use a 5 ml serological pipette to transfer 5 ml of egg water (or DMEM/F-12) into a new 15 ml conical tube labeled 'H + P'.
   1. Remove 110 μl of egg water (or DMEM/F-12) from the 15 ml conical tube labeled 'H + P' and discard.
   2. Add 10 μl of H2DCFDA working solution, then add 100 μl of PMA working solution into the 15 ml conical tube labeled 'H + P' and vortex to mix (this volume is enough for 48 samples or the other half of a full 96 well microplate).
7. Keep the dosing solutions on ice.

3. Microplate Reader Programming

1. Prepare instrument: Power on the microplate reader.
   1. Warm up the light source.
   2. Set up a program to read fluorescence: e.g. Excitation: 485 nm; Emission: 528 nm; Optics Position: top 510 nm; Sensitivity: 65, with a 5 sec shaking step prior to the read.

4. 96 Well Microplate Set Up (see Figure 2)

1. Gather supplies: Obtain dishes with dechorionated embryos, black 96 well microplate, p200 pipettor and tips, ice bucket with H2DCFDA and H2DCFDA + PMA dosing solutions, multichannel p200 pipettor, two sterile reservoirs, aluminum foil, and scissors.
2. Transfer one embryo into each well of a 96 well microplate: Use scissors to cut a pipette tip such that embryos or kidneys fit through the opening.
   1. Set a p200 pipettor to 100 μl and transfer one embryo along with egg water (or DMEM/F-12) into as many of the wells of a black 96 well microplate as desired (it is not necessary to change the pipette tip for each different embryo sample within an experimental condition, but it may be necessary to change tips between experimental conditions). Be sure to avoid transferring residual chorions, as these tend to skew the data collected. For the transfer of kidneys, a larger volume pipettor (set to 100 μl) can be used and pipette tips can be cut to obtain a larger bore size, if necessary. It may be necessary to incorporate wells without embryo or kidney samples, but with the dosing solutions to control for some experimental manipulations.
3. Add dosing solutions: Pour H2DCFDA dosing solution into a sterile 25 ml reservoir.
   1. Use a multichannel p200 pipettor and eight tips to simultaneously pipette 100 μl of H2DCFDA dosing solution into one column on the 96 well microplate (500 ng/ml final concentration of H2DCFDA).
   2. Repeat this (changing tips is not necessary) for as many columns as desired (usually six columns or 48 wells if filling an entire 96 well microplate). Add this solution to half of the control embryo samples and half of the experimentally manipulated embryo samples (an example 96 well microplate set up is shown in Figure 2, these wells (colored orange) will provide background fluorescence data in samples not induced with PMA).
   3. Pour the H2DCFDA + PMA dosing solution into a new, sterile 25 ml reservoir.
   4. Use a multichannel p200 pipettor and eight tips to simultaneously pipette 100 μl of H2DCFDA + PMA dosing solution into the remaining columns (colored red in Figure 2) of the 96 well microplate (changing tips is not necessary, final concentrations of H2DCFDA- 500 ng/ml and PMA- 200 ng/ml).
4. Cover the microplate with aluminum foil.
5. Shake the microplate for approximately 20 sec at 150 rpm to homogenize the solutions in each well.
6. Incubate the microplate at 28 °C when it is not being read.

5. Fluorescence Quantification

1. Read the microplate at time = 0 hr after the addition of PMA using the parameters described in step 3.1.2.
   1. Continue taking measurements every few minutes for the desired time interval or incubate the foil wrapped microplate at 28 °C until a later time point and then take an endpoint measurement at a desired time (e.g. 4 hr after the addition of PMA).
2. Use a plastic transfer pipette to retrieve the embryos from the wells.
3. Euthanize the embryos according to your animal care and usage protocol, e.g. immersion in tricaine MS222.
4. Dispose of the microplate and other disposable materials in the biohazardous waste container.

6. Data Analysis

1. Decide on the time point at which you would like to compare fluorescence values (e.g. 4 hr after the addition of PMA, Table 1).
2. Subtract the average un-induced control group's fluorescence value from the individual PMA-induced control group's fluorescence values.
3. Repeat this for the experimental group with and without PMA.
4. Store these normalized fluorescence values in two columns, the control + PMA group and the experimental + PMA group (Table 2).
5. Calculate the means and standard deviations for the normalized fluorescence values of the control + PMA group and the experimental + PMA group.
6. Compare the normalized fluorescence values using an unpaired t-test to determine statistical significance (Table 2).
7. Graph the means of the control + PMA group and the experimental + PMA group with error bars reflecting the appropriate standard deviations.
8. Record the level of significance on the graph and in the figure legend (Figure 2).

Wyniki

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...

Dyskusje

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...

Materiały

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