Name: assessing the cellular immune response of the fruit fly, drosophila melanogaster, using an in vivo phagocytosis assay
Uploaded: 2019-04-10T15:00:00
Description: Watch this Scientific Journal Video about Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay at JoVE.com

The authors thank Dr. Beth Gonzalez and Dr. Aprajita Garg for support in carrying out the in vivo phagocytosis experiments. An NSF UMD ADVANCE Seed Grant and UMD NIH T32 Training Grants, Cell and Molecular Biology (CMB) and the Host-Pathogen Interactions (HPI), funded this work.

1. Prepare&#160;Fluorescein particles for injection
<ol>
	<li>Reconstitute 10 mg of commercially available, heat-killed bacteria particles labeled with fluorescein (see Table of Materials) to a stock concentration of 10 mg/mL by adding 990 &#181;L sterile 1x PBS and 10 &#181;L 50 mM sodium azide. Vortex to mix.
	<ol>
		<li>Divide into single-use 8 &#181;L aliquots in 0.2 mL tubes and store in a dark box at 4 &#176;C to minimize light associated sensitivity. 
		NOTE: Sodium azide preservative is op...

<ol>
	<li>Akira, S., Uematsu, S., Takeuchi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+recognition+and+innate+immunity.">Pathogen recognition and innate immunity.</a> Cell. 124 (4), 783-801 (2006).</li><li>Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+host-pathogen+interactions+and+innate+immunity+in+Caenorhabditis+elegans.">Studying host-pathogen interactions and innate immunity in Caenorhabditis elegans.</a> Disease Models &amp; Mechanisms. 1 (4-5), 205-208 (2008).</li><li>Lemaitre, B., Hoffmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+host+defense+of+Drosophila+melanogaster.">The host defense of Drosophila melanogaster.</a> Annual Review Immunology. 25, 697-743 (2007).</li><li>Wu, L. 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Commercially available, fluorescently labeled particles are used to assess phagocytosis in general (0.2 &#181;m carboxylate-modified microspheres) or phagocytosis of microbes (fluorescently-labeled heat- or chemically killed bacteria or yeast). To assess phagosome maturation, researchers can select particles labeled with a pH-sensitive dye that fluoresces when pH decreases from neutral to acidic, as in the phagolysosome. Alternatively, to examine the initial steps of phagocytosis, pathogen recognition and up...

Innate immune defenses form the first line of defense against pathogenic microbes. These responses can be functionally divided into humoral and cellular innate immunity, both of which are mediated by germline-encoded pattern recognition receptors (PRRs) that sense pathogen-associated molecular patterns (PAMPs)1. Many of the signaling pathways and effector mechanisms of innate immunity are conserved in mammals and invertebrates, such as the nematode, Caenorhabditis elegans, and the fruit fly, Drosophila melanogaster2. The fruit fly has emerged as a powerful system to study host defense against infectious microorganisms3. Drosophila is genetically tractable, easily and inexpensively reared in laboratories, and has a short generation time. Furthermore, the fruit fly exhibits highly efficient defenses against an array of microbes, enabling the examination of host immunity against viral, bacterial, fungal, or parasitic pathogens.
Drosophila immunologists have historically utilized&#160;forward genetic screens, genome-wide RNA-mediated interference (RNAi) screening of insect cell lines, and pre-existing mutant fly strains to examine innate immunity &#8211; leading to the identification and characterization of several evolutionarily conserved humoral immune pathways4,5,6,7,8. The humoral innate immune response is, arguably, the best characterized immune defense in fruit flies. Following infection, the humoral response leads to the production and systemic release of antimicrobial peptide (AMP) molecules into the hemolymph, the blood equivalent in insects. AMPs are produced by highly conserved Toll and Imd signaling pathways. The Toll pathway is homologous to mammalian TLR/IL-1R receptor signaling, and the Imd pathway is homologous to mammalian Tumor necrosis factor-alpha signaling. In Drosophila, Toll signaling is induced by gram-positive bacteria, fungi, and Drosophila X virus6,9,10 and Imd signaling is induced by gram-negative bacteria11,12.
Cellular immunity, comprised of encapsulation, melanization, and phagocytosis of invasive pathogens,&#160;carried out by specialized blood cells called hemocytes13. There are three classes of hemocytes in the fruit fly: crystal cells, lamellocytes, and plasmatocytes13. Crystal cells, which make up 5% of the circulating hemocytes in larvae, release proPhenoloxidase (proPO) enzymes leading to the melanization of pathogens and host tissues at wound sites. Lamellocytes, which are not normally found in healthy embryos or larvae, are adherent cells that encapsulate foreign objects. These cells are induced upon pupariation or when parasitizing wasp eggs are deposited in larvae. Phagocytic plasmatocytes, which make up 95% of circulating hemocytes in larvae and all remaining hemocytes in adults, play a role in tissue remodeling during development and, notably, serve as the main effector cell of Drosophila cellular immunity.
Phagocytosis an immediate and crucial line of innate immune defense; microbes that breach the host epithelial barrier are quickly engulfed and eliminated by phagocytic blood cells (For a comprehensive review of cell biology of phagocytosis see reference 14). This process is initiated when germline-encoded pattern recognition receptors (PRRs) on hemocytes recognize pathogen associated molecular patterns (PAMPs) of microbes. Once bound to their targets, PRRs initiate signaling cascades that lead to the formation of pseudopods through actin cytoskeleton remodeling. The pseudopods surround the microbe, which is subsequently engulfed and internalized into a nascent organelle, the phagosome. Microbes are destroyed as the phagosome undergoes the process of phagosome maturation when the phagosome is trafficked towards the interior of the hemocyte and acidifies through a series of interactions with lysosomes. In vitro and cell biology studies in mammalian primary cells have been instrumental in identifying and characterizing factors that regulate phagocytosis, such as the mammalian Fc-gamma receptor and C3b receptors15,16. Nevertheless, the ability to execute large-scale screens or in vivo studies are limited in mammalian systems.
Here we present an in vivo assay for phagocytosis in adult fruit flies, which is based on a procedure first introduced by the laboratory of David Schneider in 200017. The Schneider lab showed that sessile hemocytes clustered along the abdominal dorsal vessel readily phagocytose polystyrene beads and bacteria. To visualize phagocytosis, flies are injected with fluorescently labeled particles (such as E. coli labeled with fluorescein isothiocyanate (E. coli-FITC)), incubated for 30 minutes to allow hemocytes time to engulf the particles, and then injected with trypan blue, which quenches the fluorescence of particles not phagocytosed during the incubation period. Fly dorsal vessels are then imaged using an inverted fluorescent microscope. This seminal paper, using a relatively simple experiment, demonstrated that hemocytes phagocytose bacteria and latex beads, that bacterial phagocytosis can be inhibited by pre-injecting flies with latex beads, and that flies without both cellular and humoral immune responses are susceptible even to E. coli. The assay presented in this report builds on the work of the Schneider lab to quantify in vivo phagocytosis by measuring the fluorescence intensity of particles engulfed by dorsal vessel associated hemocytes.
Similar to the approach taken in mammalian systems, Drosophila geneticists initially used genome-wide in vitro RNAi screens to identify genes required for the cellular immune response18,19,20,21,22,23. However, the development of the adult in vivo phagocytosis assay enabled follow-up experiments to be readily carried out in whole animals, thus allowing researchers to verify the biological the role of factors identified in in vitro studies. Such was the case with the transmembrane receptor Eater, which was first identified as a bacterial receptor in an RNAi screen using S2 cells24 and then later shown to mediate Escherichia coli (E. coli), Enterococcus faecalis, and Staphylococcus aureus (S. aureus) phagocytosis in adults25.
Our lab employed the in vivo phagocytosis assay in forward genetic screens and genome-wide association studies (using the Drosophila Genetic Reference Panel (DGRP)) to identify novel genes that regulate phagocytosis in adult hemocytes. These studies led to the characterization the receptors PGRP-SC1A and PGRP-SA26, the intracellular vesicle trafficking protein Rab1427, the glutamate transporter Polyphemus28, and RNA-binding protein Fox-129.
We anticipate that future screens incorporating the in vivo phagocytosis could lead to the identification of additional genes that are important for the cellular immune response in Drosophila. Screens using fully-sequenced inbred lines, such as the DGRP or the Drosophila Synthetic Population Resource (DSPR), can identify natural variants affecting phagocytosis or hemocyte development. Furthermore, the technique could be adopted in other species of Drosophila or used to screen new community resources, such as the collection of 250 Drosophila species maintained by the National Drosophila Species Stock Center (NDSSC) at Cornell. These experiments can be carried out using fluorescently-labeled bacterial or fungal-wall bioparticles that are available commercially or may be performed using any number of bacterial or fungal species &#8211; provided that the microbe expresses fluorescent markers.

<table>
	<tbody>
		<tr>
			<td>0.2&#956;m Red Fluorescent Carboxylate Modified FluoSpheres</td>
			<td>Invitrogen</td>
			<td>F8810</td>
			<td>Fluorescently-labeled latex beads to test general phagocytic capacity of phagocytes. (~580/~605 nm) Inject a 1:20 dilution in PBS with 5% dye.</td>
		</tr>
		<tr>
			<td>5430-10 PicoNozzle Kit</td>
			<td>World Precision Instruments</td>
			<td>5430-10</td>
			<td>Holder for 1.0mm pipette</td>
		</tr>
		<tr>
			<td>E. coli (K-12 Strain) BioParticles, Alexa Fluor 488 conjugate</td>
			<td>Invitrogen</td>
			<td>E13231</td>
			<td>Killed E. coli labeled with Alexa Fluor 488. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~495/~519 nm)</td>
		</tr>
		<tr>
			<td>E. coli (K-12 Strain) BioParticles, Alexa Fluor 594 conjugate</td>
			<td>Invitrogen</td>
			<td>E23370</td>
			<td>Killed E. coli labeled with Alexa Fluor 594. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~590/~617 nm)</td>
		</tr>
		<tr>
			<td>E. coli (K-12 Strain) BioParticles, Fluorescein conjugate</td>
			<td>Invitrogen</td>
			<td>E2861</td>
			<td>Killed E. coli labeled with FITC (Fluorescein). Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~494/~518 nm)</td>
		</tr>
		<tr>
			<td>E. coli (K-12 Strain) BioParticles, Texas Red conjugate</td>
			<td>Invitrogen</td>
			<td>E2863</td>
			<td>Killed E. coli labeled with Texas Red. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~595/~615 nm)</td>
		</tr>
		<tr>
			<td>E. coli (K-12 Strain) BioParticles, Texas Red conjugate</td>
			<td>Invitrogen</td>
			<td>E2863</td>
			<td>Killed E. coli labeled with Texas Red. Use to test phagocyte recogntion and uptake of gram-negative bacteria. (~595/~615 nm)</td>
		</tr>
		<tr>
			<td>Needle Pipette Puller</td>
			<td>David Kopf Instruments</td>
			<td>Model 725</td>
			<td></td>
		</tr>
		<tr>
			<td>pHrodo Red E. coli BioParticles Conjugate for Phagocytosis</td>
			<td>Invitrogen</td>
			<td>P35361</td>
			<td>Killed E. coli labeled with pHrodo Red. Use to test phagocyte reconition, uptake, and phagosome maturation of gram-negative bacteria. (~560/~585 nm). No need to quench with Trypan Blue.</td>
		</tr>
		<tr>
			<td>pHrodo Red S. aureus BioParticles Conjugate for Phagocytosis</td>
			<td>Invitrogen</td>
			<td>A10010</td>
			<td>Killed S. aureus labeled with pHrodo Red. Use to test phagocyte reconition, uptake, and phagosome maturation of gram-positve bacteria. (~560/~585 nm). No need to quench with Trypan Blue.</td>
		</tr>
		<tr>
			<td>Pneumatic PicoPump PV820</td>
			<td>World Precision Instruments</td>
			<td>SYS-PV820</td>
			<td>The World Precision Instruments Pneumatic PicoPump PV820 uses differential pressures to hold liquid in the glass needle between injections. The user manually controls short bursts of gas pressure to expel the liquid &#8211; allowing delivery of sub-nanoliter volumes. The amount of liquid delivered depends on two main variables &#8211; the size of the glass needle opening and the amount of time injection pressure is applied. set the instrument to 100 ms &#8220;TIMED&#8221; mode.</td>
		</tr>
		<tr>
			<td>S. aureus (Wood Strain without protein A) BioParticles, Alexa Fluor 488 conjugate</td>
			<td>Invitrogen</td>
			<td>S23371</td>
			<td>Killed S. aureus labeled with Alexa Fluor 488. Use to test phagocyte recogntion and uptake of gram-positive bacteria. (~495/~519 nm)</td>
		</tr>
		<tr>
			<td>S. aureus (Wood Strain without protein A) BioParticles, Alexa Fluor 594 conjugate</td>
			<td>Invitrogen</td>
			<td>S23372</td>
			<td>Killed S. aureus labeled with Alexa Fluor 594. Use to test phagocyte recogntion and uptake of gram-positive bacteria. (~590/~617 nm)</td>
		</tr>
		<tr>
			<td>S. aureus (Wood Strain without protein A) BioParticles, Fluorescein conjugate</td>
			<td>Invitrogen</td>
			<td>E2851</td>
			<td>Killed S. aureus labeled with FITC (Fluorescein). Use to test phagocyte recogntion and uptake of gram-positive bacteria. (~494/~518 nm)</td>
		</tr>
		<tr>
			<td>Thin Wall Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>TW100F-3</td>
			<td>Needles for injection. OD = 1.0 mm</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution (0.4%)</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>Used to quench extracellular fluorescence of Fluorescein, Alexa Fluor, or Texas Red labeled particles.</td>
		</tr>
		<tr>
			<td>ZEISS SteREO Microscope (Discovery.V8)</td>
			<td>Zeiss</td>
			<td>SteREO Discovery.V8</td>
			<td>Inverted fluorescence microscope for imaging flies. Use a digital camera (example: AxioCam HC camera) and the accompanying software (example: AxioVision 4.7 software) to take pictures.</td>
		</tr>
		<tr>
			<td>Zymosan A (Saccharomyces cerevisiae) BioParticles, Alexa Fluor 488 conjugate</td>
			<td>Invitrogen</td>
			<td>Z23373</td>
			<td>Killed labeled with Alexa Fluor 488. Use to test phagocyte recogntion and uptake of yeast. (~495/~519 nm)</td>
		</tr>
		<tr>
			<td>Zymosan A (Saccharomyces cerevisiae) BioParticles, Alexa Fluor 594 conjugate</td>
			<td>Invitrogen</td>
			<td>Z23374</td>
			<td>Killed labeled with Alexa Fluor 594. Use to test phagocyte recogntion and uptake of yeast. (~590/~617 nm)</td>
		</tr>
		<tr>
			<td>Zymosan A (Saccharomyces cerevisiae) BioParticles, Fluorescein conjugate</td>
			<td>Invitrogen</td>
			<td>Z2841</td>
			<td>Killed labeled with FITC (Fluorescein). Use to test phagocyte recogntion and uptake of yeast. (~494/~518 nm)</td>
		</tr>
		<tr>
			<td>Zymosan A (Saccharomyces cerevisiae) BioParticles, Texas Red</td>
			<td>Invitrogen</td>
			<td>Z2843</td>
			<td>Killed labeled with Texas Red. Use to test phagocyte recogntion and uptake of yeast. (~595/~615 nm)</td>
		</tr>
	</tbody>
</table>

assessing the cellular immune response of the fruit fly, drosophila melanogaster, using an in vivo phagocytosis assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

A schematic of the in vivo phagocytosis assay using fluorescein-labeled particles is shown in Figure 1A. Flies are mounted ventral side down on a piece of electrical tape and the first two segments of the abdomen, where the dorsal vessel is located, is clearly visible (Figure 1B). Key sources of experimental error arise at the injection and imaging steps of the procedure (Figure 1C). Using the same n...

Watch this Scientific Journal Video about Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay at JoVE.com

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

This in vivo phagocytosis assay allows researchers to carry out genetic screens and genome-wide association studies to identify novel genes that regulate phagocytosis in adult blood cells. This experiment is quantitative, easy to perform, and can be applied to the screening of live animals for host factors that influence pathogen recognition, uptake, and clearance. After pulling thin-wall glass capillaries with a needle puller, use a micrometer to hold the needle under a microscope, and use number five, fine-point, stainless-steel tweezers to break the tip to a 100-micrometer tip diameter.To measure the volume of liquid that will be injected into each fly, load a capillary needle with sterile 5%food coloring in PBS, and expel the liquid onto a drop of mineral oil on a 0.01-millimeter stage micrometer. Dispense 10 microliters of 1.6 milligrams per milliliter particles onto a small square of Parafilm, and pull the liquid into the needle. Mount the needle into the injector nozzle, and line the anesthetized flies along their designated area on the fly pad, ventral side up, with the heads oriented toward the front of the pad.Place the vials in corresponding areas on the bench, and inject the flies at the upper corner of the abdomen with five, 100-millisecond pumps of liquid to deliver about 10 nanoliters of particles total. Transfer each fly into the appropriate vial as it is injected, noting the time on the vial. Next, load a new needle with 0.4%Trypan Blue solution, and set the pneumatic injector to gated, to allow a constant flow of air to push the liquid out of the needle.30 minutes after the initial injection, inject each fly abdomen with Trypan Blue until the abdomens are full and distended. Mount the flies on microscope slides with electrical tape, ventral side down, pushing the wings to the side of the fly to secure them to the tape. Then, gently push the head into the tape to ensure that the fly will not move.Immediately after all of the flies have been secured, image the insects, one at a time, at a 25 or 32 times magnification on an inverted fluorescence microscope attached to a digital camera and computer, focusing on the dorsal vessel of each fly using the computer software for the digital camera. Then, record the exposure time and magnification between experiments. To quantify the fluorescence, open an appropriate imaging analysis program, and open one image.To measure the fluorescence intensity of the dorsal vessel, draw a polygon around the dorsal vessel, and select Measure to record the fluorescence intensity inside the polygon. To determine the background fluorescence intensity, copy the first polygon, and move it to an area adjacent to the dorsal vessel of each fly. Then, select Measure, and record the fluorescence intensity of the background area.To normalize the dorsal vessel fluorescence by the background fluorescence, after measuring the fluorescence intensities of the rest of the flies, divide the dorsal vessel fluorescence by the background fluorescence, and calculate the average normalized dorsal vessel fluorescence intensity of all the flies in one strain. After being mounted ventral side down on a piece of electrical tape, the first two segments of the abdomen, where the dorsal vessel is located, are clearly visible. Key sources of experimental error arise at the injection and imaging steps of the procedure.Using the same needle to inject multiple flies may cause it to become clogged with fly tissue or particles. Flies that do not receive enough Trypan Blue fluorescence brightly throughout their entire abdomen, which can reduce the ratio of the dorsal vessel to the background fluorescence, thus decreasing the true fluorescence intensity ratio of the animal. Flies should be photographed while immobilized by carbon dioxide, as actively moving flies produce blurry images that cannot be quantified.A mutant line from the Zuker collection of ethyl methanesulfonate-treated flies are unable to phagocytose gram-negative and gram-positive bacteria and displays almost no dorsal vessel fluorescence in the in vivo phagocytosis assay, paired to the isogenic background Zuker strain and another common laboratory control strain. Keep track of the time and the order in which flies are injected to ensure that flies are at comparable stages of pathogen recognition and uptake when imaged. The molecular mechanisms underlying phagocytic defects can be determined by studying single hemocytes with confocal microscopy or after fluorescence-activated cell sorting or by magnetic bead isolation of adult hemocytes.

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