This work was supported by the National Science Foundation under grant #IOS-1025627 and by the National Institutes of Health under grant #1R21NS078582-01 to J.A.W.

This protocol uses two setups (Figure 1) to acquire four different readouts collected from flies subjected to a bacterial infection. These outputs include 1) sleep/wake behavior; 2) survival outcome; 3) bacterial load in the fly; and 4) real-time measurement of NF&kappa;B reporter activity in vivo. In combination with the genetic tools that are available in Drosophila, these measurements provide mechanistic insight into the molecular link between immune function and behavior.
1. Measure Locomotor Activity and Sleep in Flies
<ol>
	<li>The setup used to measure locomotor activity and sleep in flies, which includes the Drosophila activity monitoring system (DAM2, Trikinetics), incubators, dark room, and the preparation of experimental animals and activity tubes, has been described previously6. The same approach is used here, with some minor modifications.
	<ol class="lalpha">
		<li>The lighting of the incubator is controlled by the incubator itself. Therefore, it is important to synchronize the time setting among computer and incubators.</li>
		<li>Activity tubes are cleaned for re-use by boiling on a hot plate in de-ionized water. A small amount of detergent is added for the first boil, tubes are well-rinsed and boiled twice more. If yarn is used to plug the tube, used activity tubes (containing food, wax, fly and yarn) can be cleaned without prior removal of the yarn.</li>
	</ol>
	</li>
	<li>Prior to initiating an experiment, place cultures containing late pupal staged flies in incubators for three to four days to adapt to experimental lighting and other environmental conditions. Light:dark or constant conditions have been commonly used to measure sleep behavior. In this example, flies are acclimated to constant light to eliminate the influence of the circadian clock on the immune response and behavior2,7,8 . As described in Figure 1A, load 1-4 day old flies into the Trikinetics DAM2 activity monitors as described6, and record for a minimum of three days prior to the infection.</li>
</ol>
2. Infect Flies with a Pathogenic Strain of Bacteria
<ol>
	<li>The protocol described here is specific for S. marcescens, which are easy to grow and maintain. Protocols for long-term storage and culture conditions for other bacterial species will vary. S. marcescens are stored in 15-50 % glycerol at -80 &deg;C. To prepare for long-term storage, mix 2 volumes of overnight bacterial culture (O.D. 600 = 0.5 - 1.0) to 1 volume of autoclaved 50% glycerol in 1.5 ml microcentrifuge tubes. This will result in a final glycerol concentration of 17%. Store tubes at -80 &deg;C. To prepare a short-term in-use source of bacteria, scrape the frozen stock with a sterile 200 &mu;l pipette tip and perform a three-way streak on an LB agar plate. Incubate the plate overnight at 37 &deg;C to get isolated colonies. Store the plate at 4 &deg;C. 
	One day before the scheduled infection, pick a single colony from the LB agar plate with a sterile 200 &mu;l pipette tip and submerge the tip into a culture tube containing 5 ml LB medium. Grow bacteria overnight, or up to 16 hr inside an incubator shaker at 37 &deg;C and 250 rpm until it reaches the exponential growth phase. Measure the concentration with a spectrophotometer at OD600. The concentration in this phase ranges from 0.5 to 1. If the concentration is too high, subculture the bacteria and grow for another several hours to get an optimal concentration. Perform the procedure near a flame source. Some bacterial strains are engineered for antibiotic resistance and are grown and selected for appropriate antibiotic added to the medium. S. marcescens (ATCC #8100) are not antibiotic resistant and are therefore grown in sterile medium without antibiotic. To verify sterile technique, do a mock culture without bacteria as a control. 
	The solution to infect flies contains bacteria (diluted to OD600=0.1) and 1% food coloring (Brilliant Blue FCF) in PBS. Prepare a solution for injection control by adding equivalent amount of LB medium used in infection solution and 1% blue food coloring in PBS. Store solutions on ice.</li>
	<li>Prepare glass needles for injecting the flies. Pull glass capillaries (o.d. = 1 mm, i.d. = 0.58 mm, WPI) to a fine tip using a micropipette puller (Narishige). Under a dissecting microscope, use fine forceps to break off the tip of the needle so that the opening is large enough to fill with injection fluid by applying suction with a syringe, but small enough to minimize damage to the fly during the injection process. After breaking the tip, the tip size should be around 40-50 &mu;m. The glass needle is attached to a 3 cc plastic syringe with a length of tubing. The flow of injection medium is controlled manually applying positive pressure or suction with the syringe. Avoid contaminating the rubber tubing with injection fluid, as the syringe apparatus is used for both infection and control injections.</li>
	<li>Anesthetize flies by putting them on a CO2 pad. CO2 is passed through a sealed container of water to humidify the pad and to reduce static electricity, which can complicate manipulation of flies on the pad. Inject flies by poking the glass needle into the region above the scutellum of the dorsal thorax. The passing of injection medium into the fly is verified by the food coloring - flies turn blue as the food coloring spreads through the system. The dose of bacteria that flies receive can be quantified as described in Section 3, below. Some experimental designs include a control group that receives aseptic injury by injecting flies with PBS and food coloring but without bacteria.</li>
</ol>
3. Determine the Bacterial Load
One approach to evaluating the immune response against bacterial infection is to determine the bacterial load post infection. D. melanogaster is a great model to determine this parameter because the whole fly can be homogenized to estimate the total bacterial numbers within an individual. The rationale behind this protocol is that when grown on a solid medium such as Luria broth (LB) agar on a Petri dish (LB plate), a single bacterium forms a visible distinguishable colony. Therefore, by homogenizing infected flies in LB liquid medium, generating serial dilutions of the homogenate, and spreading the diluted homogenate onto LB plates, the number of bacterial cells infecting a fly can be determined. A control group of flies injected with PBS and food coloring but without bacteria should be used to verify that the infection was not contaminated with other bacterial species. There should be no colonies on the LB agar plate in this condition.
<ol>
	<li>Autoclave all materials that will be used for this procedure before performing the actual homogenization step. This includes 200 &mu;l pipette tips cut with scissors, LB media, and pestles used for the homogenization. Prepare plates by pouring autoclaved LB/agar medium into 10 cm sterile Petri dishes at least 1 day before homogenizing flies.</li>
	<li>Anesthetize and collect flies in 1.5 ml microcentrifuge tubes and store tubes on ice.</li>
	<li>Perform the homogenization near a flame to prevent contamination. A control homogenization containing flies without infection is recommended especially when using a bacterial strain such as S. marcescens, which are not antibiotic resistant. Homogenize a minimum of 2 groups of 10 flies each per experimental condition. The number of flies used per homogenate is determined by the experimenter. Some groups have used one fly per homogenate, which is useful for evaluating variation in bacterial load between individual flies8, while others have used from 3-10 flies per homogenate to compare across genotype or experimental condition9-12.
	<ol class="lalpha">
		<li>Add 400 &mu;l LB medium to each microcentrifuge tube containing flies and homogenize using a small motor and pestle (Kontes).</li>
		<li>Using sterilized cut pipette tips, serial dilute the homogenate 1:10 by adding 20 &mu;l homogenized LB/bacterial medium to 1.5 &mu;l microcentrifuge tube containing 180 &mu;l LB medium. Cutting pipette tips prevents blockage from fly debris and ensures that an appropriate volume is being transferred.</li>
		<li>Dilution factor is determined empirically and depends on the genotype of the fly, the bacterial strain used, and experimental condition. For wild type flies infected with S. marcescens, use dilutions of 1:102 or 1:103 for flies homogenized immediately post-infection, and dilutions of 1:104 or 1:105 for flies homogenized 24 hr post-infection.</li>
		<li>Add 100 &mu;l LB/bacterial medium from the microcentrifuge tubes containing the final dilution and spread the medium on an LB plate using glass balls (VWR) to ensure an even distribution.</li>
		<li>Discard the glass balls into 100% EtOH solution. Place the LB plates in a 37 &deg;C incubator overnight.</li>
		<li>As an optional step, use an imaging system with visual light to obtain an image of the LB plates (FluorChem 8900; Alpha Innotech). Count the numbers of colonies either on the plate itself or from the image of the plate using the counting tool on Photoshop software (Adobe Photoshop CS3).</li>
		<li>Calculate the number of colony-forming units per fly using the formula: [n /(N *D *v)]*V; where n= number of colonies grown on the LB plate; N=the number of the flies pooled in one microcentrifuge tube; D= dilution factor; and v = volume of solution spread onto each plate; V = initial volume of the homogenate.</li>
	</ol>
	</li>
</ol>
4. Evaluate Sleep and Survival Duration After Infection
<ol>
	<li>For flies that are not harvested for measuring bacterial load, continue monitoring behavior for another 7-10 days. Survival duration is influenced by genotype of the fly, environmental conditions, and bacterial species used for infection.</li>
	<li>After ~10 days, terminate the experiment, download and process behavioral data as previously described6. Insomniac2 custom software, which is based in Matlab, is used to analyze sleep parameters. It is important to eliminate dead flies from the behavioral analysis. Normally, this restricts analyses to within the first day after infection, depending on the type of infection and fly genotype. Death in the assay is indicated by the time all activity counts have reached zero. To facilitate analysis of survival, Drosonex, custom software written in Microsoft Visual C++ 6.0, is used to process raw data files from the Trikinetics DAM system. The software reports as Excel files, survival duration of each fly (in hours), compiles activity data from all monitors into a single spreadsheet, and reports the percent flies surviving over time as set by the user. The Excel files are designed to integrate into other statistical software packages for further analysis.</li>
</ol>
5. Measure NF&kappa;B Activity During Infection Using a Luciferase Reporter Assay
Transgenic &kappa;B-luc flies used in this assay were generated previously as described in Kuo et al., 20102. Briefly, the &kappa;B-luc reporter contains 8 repeats of an NF&kappa;B binding sequence that were inserted into a promoter upstream of a luciferase open reading frame.
<ol>
	<li>Adjust flies to experimental lighting conditions as described in section 1.2. In this example, 1-4 day old &kappa;B-luc flies are housed in vials containing a 5% sucrose, 2% agar food medium and placed in constant light (LL) for 2 days to reach the same age as other flies during infection.</li>
	<li>Prepare a 96-well microplate for flies. Each well contains 2 layers of food medium (Figure 2); the top layer contains luciferin, the substrate of luciferase. Add 300 &mu;l of a 5% sucrose, 2% agar solution to each well and allow to solidify. Next, add a 50 &mu;l top layer to each well containing 5% sucrose, 1% agar, and 2 mM luciferin (Gold BioTechnology, Inc.). Luciferin concentrations used for measuring reporter activity in Drosophila have varied in the literature, and have been as low as 100 &mu;M13. The concentration required can be determined empirically. Between dispensing the food layers, cover the plate with a fine mesh cloth to facilitate drying while maintaining sterility. The plate should be allowed to dry thoroughly, up to one hour, in order to prevent flies from getting stuck in condensation droplets. Because luciferin is light-sensitive, avoid exposing plates to light more than necessary.</li>
	<li>Apply a clear adhesive film (Top-Seal-A; Perkin Elmer) to a 96-well microwell plate and perforate using a fine needle at a quantity of 2 holes per well. These holes will not only allow air exchange to each well, but will also provide a way to anesthetize flies on an individual basis. Using a sharp blade and straight edge, introduce a cut between each column. This will provide an easy way to load/unload flies in groups of 8 at a time. 
	To load flies into the microwell plate, remove the vials from the incubator and anesthetize flies on a CO2 pad. Load flies one-by-one to each well column-by-column. Should a fly inadvertently get stuck to the adhesive seal, leave the fly alone as they are often able to free themselves without intervention. Return the microwell plate to the incubator in LL for 8-24 hr to adjust to the new environment and to consume the luciferin substrate.</li>
	<li>Culture bacteria, S. marcescens, and prepare a solution for infection as described above.</li>
	<li>Anaesthetize the flies by using a micropipette tip attached to a low-pressure CO2 line. Place the micropipette tip directly above the ventilation holes made for each well. Take caution to ensure that the CO2 pressure is high enough to anaesthetize the fly, but low enough to avoid injury to the fly. In groups of eight, individually transfer each fly to a CO2 pad, and infect as described above. After infection, return each fly to its original well and reseal the microplate.</li>
	<li>Measure luminescence (TopCount-NXT Luminescence and Scintillation Counter; Perkin-Elmer). The TopCount luminescence counter contains a stacking cassette for plates, which allows for programmed and automated readings continuously at desired increments for any length of time (usually up to five days). This feature is not available on all instruments, and data collection for experiments performed with other instruments may therefore be less frequent. The luminescence counter is housed in a room with a controlled temperature and lighting schedule. When loading plates into the stacking cassette, stack the plates containing the flies between clear blank plates to ensure that flies receive light. Program the luminometer according to manufacturer's specifications to collect readings every hour for a minimum of 24 hr. In this example, the detectors are programmed to read each well for 10 sec and to express the result as counts (arbitrary units) per second. This value is averaged across 3 readings per well. Export the data files to a spreadsheet and perform a standard analysis, graphing the results of luminescence.</li>
</ol>

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No conflicts of interest declared.

This protocol outlines an approach to investigate how behavior, particularly sleep, is linked to immune response parameters. These parameters include bacterial load, survival outcome, and NF&kappa;B activity as measured by a luciferase reporter in vivo. Together these parameters provide information about how well a fly can fight an infection. Bacterial load and survival outcome are immune response parameters that involve a straightforward measurement in Drosophila. RelE20 muta...

<table border="1">
 <tbody>
 <tr>
 <td>Material Name </td>
 <td>Company </td>
 <td>Catalogue number </td>
 <td>Comments </td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>Incubators</td>
 <td>Percival Scientific, Inc.</td>
 <td>I30BLLC8 
 I36VLC8</td>
 <td>Any incubator capable of running programmed light/temperature schedules is appropriate.</td>
 </tr>
 <tr>
 <td>Drosophila Activitiy Monitors</td>
 <td>Trikinetics Inc., Waltham, MA</td>
 <td>DAM2</td>
 <td>As described elsewhere6, this system requires a computer interface, software, and other accessories.</td>
 </tr>
 <tr>
 <td>Pyrex Glass Tubes</td>
 <td>Trikinetics Inc., Waltham, MA</td>
 <td>PGT-5x65</td>
 <td></td>
 </tr>
 <tr>
 <td>Microplate scintillation and luminescence counter</td>
 <td>Perkin Elmer</td>
 <td>TopCount NXT 
 12 detector </td>
 <td>Any microplate reader capable of detecting luminescence can be used for this type of reporter assay. TopCount contains multiple detectors and an automated stacker; it is capable of being programmed to read continuously from multiple plates. </td>
 </tr>
 <tr>
 <td>FluorChem 8900</td>
 <td>Alpha Innotech</td>
 <td></td>
 <td>Imaging of bacterial cultures is optional; any digital imaging system with visual light capability is sufficient.</td>
 </tr>
 <tr>
 <td>Micropipette Puller</td>
 <td>Tritech Research, Inc.</td>
 <td>Narishige PC-10</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Supplies </td>
 </tr>
 <tr>
 <td>Borosilicate Glass Capillaries</td>
 <td>World Precision Instrument Inc.</td>
 <td>1B100F-4</td>
 <td></td>
 </tr>
 <tr>
 <td>3 ml Syringe</td>
 <td>Fisher Scientific</td>
 <td>BD 305482</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe Needles</td>
 <td>Fisher Scientific</td>
 <td>BD 305196</td>
 <td>18 G - cut off the tip of the needle to prevent damage to the tubing. </td>
 </tr>
 <tr>
 <td>Silicone Tubing, i.d. (0.030") o.d. (0.065") Wall Thickness (0.018")</td>
 <td>VWR</td>
 <td>60985-706</td>
 <td>Used for attaching glass capillary needles to a syringe</td>
 </tr>
 <tr>
 <td>3 Way Stopcock</td>
 <td>American Pharmaseal Company</td>
 <td>K75</td>
 <td></td>
 </tr>
 <tr>
 <td>Kontes Pellet Pestle Cordless Motor</td>
 <td>Fisher Scientific</td>
 <td>K749540-0000</td>
 <td></td>
 </tr>
 <tr>
 <td>Kontes Pellet Pestle </td>
 <td>Fisher Scientific </td>
 <td>K749521-1590</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass balls 3mm</td>
 <td>VWR</td>
 <td>26396-630</td>
 <td></td>
 </tr>
 <tr>
 <td>Microplate Microlite 1+</td>
 <td>Thermo Scientific</td>
 <td>7571</td>
 <td>Select 96-well plates that are appropriate for luminescence - they must be opaque.</td>
 </tr>
 <tr>
 <td>TopSeal-A:96-well Microplates</td>
 <td>PerkinElmer</td>
 <td>6005185</td>
 <td>Microplate Press-On Adhesive Sealing Film</td>
 </tr>
 <tr>
 <td>D-Luciferin, Potassium Salt</td>
 <td>Gold BioTechnology, Inc.</td>
 <td>LUCNA</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Software </td>
 </tr>
 <tr>
 <td>Insomniac2</td>
 <td>Available upon request to the authors</td>
 <td>custom; written by Lesley Ashmore, Ph.D. (Westminster College) </td>
 <td>Matlab based software that has been used routinely for analysis of sleep2,6,11 </td>
 </tr>
 <tr>
 <td>Drosonex</td>
 <td>Available upon request to the authors</td>
 <td>custom; written by Thomas Coradetti (Sidewalk Software) </td>
 <td>A PC MSVC6 program used for survival analysis from raw data files collected with the Trikinetics system</td>
 </tr>
 <tr>
 <td>Photoshop CS3</td>
 <td>Adobe</td>
 <td></td>
 <td>Useful for obtaining numbers of cfu/plate from digital images (optional)</td>
 </tr>
 </tbody>
</table>

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.

<ol>
	<li>Infection promotes sleep. In this example, Canton-S (CS) wild type flies and mutant flies lacking an NF&kappa;B gene, Relish (RelE20) 14, were loaded into two DAM2 activity monitors (n=32 for each genotype) and infected as described above. Flies were maintained in constant light to eliminate the influence of the circadian clock on behavior and infection2,7,8. The RelE20 mutants were isogenized to CS as described previously11. Both sets of flies were infe...

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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.

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 ...

quantitative-measurement-immune-response-sleep

Research

JoVE Journal

Immunology and Infection

15.3K Views.  University of Pennsylvania Perelman School of Medicine. لفهم العلاقة بين الاستجابة المناعية والسلوك، ونحن تصف طريقة لقياس السلوك الحركي في ذبابة الفاكهة خلال العدوى البكتيرية وكذلك قدرة الذباب على شن استجابة مناعية من البقاء على قيد الحياة الرصد والحمل البكتيري، في الوقت الحقيقي ونشاط منظم الرئيسية المناع...

Video: Quantitative Measurement of the Immune Response and Sleep in Drosophila

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

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. ...

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.

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). ...

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

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 ...

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

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 ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...