The authors would like to acknowledge past and present members of the Kim laboratory, Mark Nilan for zebrafish care and maintenance, Dr. Robert Wheeler for helpful discussions and data sharing, and NIH grants 3RO1GM087308-02S1 and 1P20RR024475-01A2 and the Maine Agricultural and Forest Experiment Station (Publication Number 3303) for funding.

1. Zebrafish Care and Maintenance
<ol>
	<li>Husbandry: Mass spawn adult zebrafish as previously described13. Collect spawned embryos as previously described14.</li>
	<li>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.
	<ol>
		<li>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).</li>
	</ol>
	</li>
	<li>Maintain embryos: Grow embryos in deep petri dishes at 28 &#176;C in egg water (60 &#956;g/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2-thiourea (PTU) or golden/slc24a5 mutant zebrafish does not significantly alter the induction of fluorescence by PMA (unpublished data, embryos tested at 48, 72, and 96 hpf).
	<ol>
		<li>Remove dead embryos daily with a plastic transfer pipette.</li>
		<li>Carefully decant old egg water and replenish with new egg water daily.</li>
	</ol>
	</li>
	<li>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 described16,17).</li>
</ol>
2. Solution Preparation
<ol>
	<li>Prepare a stock solution of H2DCFDA: Weigh out 1 mg of H2DCFDA.
	<ol>
		<li>Dissolve 1 mg of H2DCFDA in 1 ml of dimethyl sulfoxide (DMSO) to make a 1 mg/ml stock solution.</li>
		<li>Make 22 &#956;l aliquots of this stock solution in 1.7 ml microcentrifuge tubes.</li>
		<li>Wrap the aliquots of H2DCFDA stock solution in aluminum foil and keep in the dark whenever possible because H2DCFDA is light sensitive.</li>
		<li>Store H2DCFDA aliquots at -20 &#176;C for up to 3 months.</li>
	</ol>
	</li>
	<li>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).
	<ol>
		<li>Dissolve PMA in 1 ml of DMSO to make a 1 mg/ml stock solution.</li>
		<li>Make 11 &#956;l aliquots in 1.7 ml microcentrifuge tubes.</li>
		<li>Store aliquots of PMA stock solution at -80 &#176;C for up to 3 months.</li>
	</ol>
	</li>
	<li>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 &#956;g/ml H2DCFDA final concentration). For example, add 20 &#956;l of H2DCFDA stock solution and 20 &#956;l DMSO to a foil wrapped 1.7 ml microcentrifuge tube.</li>
	<li>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 &#956;g/ml PMA final concentration). For example, dilute 10 &#956;l PMA stock solution in 490 &#956;l nuclease free water in a 1.7 ml microcentrifuge tube.</li>
	<li>Prepare a dosing solution of H2DCFDA: On the day of the experiment, make a H2DCFDA dosing solution with a final concentration of 1 &#956;g/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&#39;s modified Eagle&#39;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 &#39;H&#39;.
	<ol>
		<li>Remove 10 &#956;l of egg water (or DMEM/F-12) from the 15 ml conical tube labeled &#39;H&#39; and discard.</li>
		<li>Add 10 &#956;l of H2DCFDA working solution into the 15 ml conical tube labeled &#39;H&#39; 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).</li>
	</ol>
	</li>
	<li>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 &#956;g/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 &#39;H + P&#39;.
	<ol>
		<li>Remove 110 &#956;l of egg water (or DMEM/F-12) from the 15 ml conical tube labeled &#39;H + P&#39; and discard.</li>
		<li>Add 10 &#956;l of H2DCFDA working solution, then add 100 &#956;l of PMA working solution into the 15 ml conical tube labeled &#39;H + P&#39; and vortex to mix (this volume is enough for 48 samples or the other half of a full 96 well microplate).</li>
	</ol>
	</li>
	<li>Keep the dosing solutions on ice.</li>
</ol>
3. Microplate Reader Programming
<ol>
	<li>Prepare instrument: Power on the microplate reader.
	<ol>
		<li>Warm up the light source.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. 96 Well Microplate Set Up (see Figure 2)
<ol>
	<li>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.</li>
	<li>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.
	<ol>
		<li>Set a p200 pipettor to 100 &#956;l 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 &#956;l) 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.</li>
	</ol>
	</li>
	<li>Add dosing solutions: Pour H2DCFDA dosing solution into a sterile 25 ml reservoir.
	<ol>
		<li>Use a multichannel p200 pipettor and eight tips to simultaneously pipette 100 &#956;l of H2DCFDA dosing solution into one column on the 96 well microplate (500 ng/ml final concentration of H2DCFDA).</li>
		<li>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).</li>
		<li>Pour the H2DCFDA + PMA dosing solution into a new, sterile 25 ml reservoir.</li>
		<li>Use a multichannel p200 pipettor and eight tips to simultaneously pipette 100 &#956;l 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).</li>
	</ol>
	</li>
	<li>Cover the microplate with aluminum foil.</li>
	<li>Shake the microplate for approximately 20 sec at 150 rpm to homogenize the solutions in each well.</li>
	<li>Incubate the microplate at 28 &#176;C when it is not being read.</li>
</ol>
5. Fluorescence Quantification
<ol>
	<li>Read the microplate at time = 0 hr after the addition of PMA using the parameters described in step 3.1.2.
	<ol>
		<li>Continue taking measurements every few minutes for the desired time interval or incubate the foil wrapped microplate at 28 &#176;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).</li>
	</ol>
	</li>
	<li>Use a plastic transfer pipette to retrieve the embryos from the wells.</li>
	<li>Euthanize the embryos according to your animal care and usage protocol, e.g. immersion in tricaine MS222.</li>
	<li>Dispose of the microplate and other disposable materials in the biohazardous waste container.</li>
</ol>
6. Data Analysis
<ol>
	<li>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).</li>
	<li>Subtract the average un-induced control group&#39;s fluorescence value from the individual PMA-induced control group&#39;s fluorescence values.</li>
	<li>Repeat this for the experimental group with and without PMA.</li>
	<li>Store these normalized fluorescence values in two columns, the control + PMA group and the experimental + PMA group (Table 2).</li>
	<li>Calculate the means and standard deviations for the normalized fluorescence values of the control + PMA group and the experimental + PMA group.</li>
	<li>Compare the normalized fluorescence values using an unpaired t-test to determine statistical significance (Table 2).</li>
	<li>Graph the means of the control + PMA group and the experimental + PMA group with error bars reflecting the appropriate standard deviations.</li>
	<li>Record the level of significance on the graph and in the figure legend (Figure 2).</li>
</ol>

<ol>
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W., Zakrzewska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+Profiling+and+Functional+Analyses+of+the+Zebrafish+Embryonic+Innate+Immune+Response+to+Salmonella+Infection.">Transcriptome Profiling and Functional Analyses of the Zebrafish Embryonic Innate Immune Response to Salmonella Infection.</a> J Immunol. 9. 9, 5641-5653 (2009).</li><li>Ordas, A., Hegedus, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+Sequencing+of+the+Innate+Immune+Transcriptomic+Response+of+Zebrafish+Embryos+to+Salmonella+Infection.">Deep Sequencing of the Innate Immune Transcriptomic Response of Zebrafish Embryos to Salmonella Infection.</a> Fish &amp; Shellfish Immunology. 31, 716-724 (2011).</li><li>Renshaw, S. A., Loynes, C. 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(NIH R01 RR15402).</a> ZFIN Direct Data Submission. , (2001).</li><li>Thisse, B., Thisse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+Release+Clones:+A+High+Throughput+Expression+Analysis.">Fast Release Clones: A High Throughput Expression Analysis.</a> ZFIN Direct Data Submission. , (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Table+18.4.">Table 18.4.</a> The Molecular Probes Handbook. , .</li></ol>

The authors have nothing to disclose.

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...

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 immunity3,4. 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 promoter5,6 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 pathogens8,9. 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&#39;,7&#39;-dichlorodihydrofluorescein diacetate (H2DCFDA), is converted into the fluorescent compound, 2&#39;,7&#39;-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.

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

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.

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...

Watch this Scientific Journal Video about Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish at JoVE.com

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

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.

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

quantification-respiratory-burst-response-as-an-indicator-innate

Research

JoVE Journal

Immunology and Infection

21.6K Views.  University of Maine.  La risposta immunitaria innata protegge contro l'infezione da organismi patogeni. Una componente fondamentale della risposta immunitaria innata, il burst respiratorio dei fagociti, genera specie reattive dell'ossigeno che uccidono i microrganismi invasori. Descriviamo un saggio burst respiratorio che quantifica specie reattive dell'ossigeno prodotte quando la risposta immunitaria innata è indotta chimicamente. 

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

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...