Name: targeting drugs to larval zebrafish macrophages by injecting drug-loaded liposomes
Uploaded: 2020-02-18T13:00:00
Description: Watch this Scientific Journal Video about Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes at JoVE.com

This work was supported by grants awarded to C.J.H. (Health research Council of New Zealand and Marsden Fund, Royal Society of New Zealand) and Z.W. (Faculty Research Development Fund from the University of Auckland). The authors thank Alhad Mahagaonkar for expert management of the zebrafish facility, the Biomedical Imaging Research Unit, School of Medical Sciences, University of Auckland for assistance with confocal imaging and Graham Lieschke for gifting the Tg(mpeg1:EGFP) reporter line.

1. Preparation of Drug-loaded Marina Blue-labeled Liposomes
NOTE: Liposomes carrying the blue fluorescent dye, Marina Blue and drug are prepared using a thin film hydration method with post insertion of poloxamer 188. All procedures are performed at room temperature unless otherwise specified. Control liposomes only carry Marina blue and PBS. The example here describes loading liposomes with a mitochondria-targeting antioxidant drug25 that is used in the representative re...

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Here, we have provided a detailed protocol to formulate drug-loaded liposomes to specifically target macrophages in larval zebrafish. This method can be used to dissect the role of macrophages in certain disease models by ensuring direct targeted delivery of drugs specifically to macrophages. Moreover, it can be used when general toxicity of drugs limits their use when delivered by more conventional routes, like immersion. The protocol described here provides an alternative to other nanoparticulate systems that have been...

The mononuclear phagocyte system provides a first line of defense against invading pathogens. This system consists of monocytes, monocyte-derived dendritic cells and macrophages, which actively phagocytoze foreign pathogens, thereby limiting pathogen spread. In addition to these phagocytic and microbicidal effector functions, dendritic cells and macrophages are also capable of cytokine production and antigen-presentation to activate the adaptive immune system1. Of these cells, macrophages have received particular attention due to their diverse functional heterogeneity and involvement in multiple inflammatory diseases, from autoimmunity and infectious diseases to cancer2,3,4,5,6,7. The plasticity of macrophages and their ability to functionally adapt to the needs to their tissue environment necessitates experimental approaches to directly observe and interrogate these cells in vivo.
Larval zebrafish are an ideal model organism by which to study the function and plasticity of macrophages in vivo8. The optical transparency of larval zebrafish provides a window to directly observe the behavior of macrophages, especially when coupled with macrophage-marking transgenic reporter lines. Exploiting the live imaging potential and experimental tractability of larval zebrafish has led to many significant insights into macrophage function that have direct relevance to human disease9,10,11,12,13,14,15. Many of these studies have also taken advantage of the high conservation of drug activity in zebrafish (an attribute that underpins their use as a whole animal drug discovery platform16,17,18), by utilizing chemical interventions to pharmacologically manipulate macrophage function. To date, these pharmacological treatments have been mostly delivered either through immersion, which requires the drug to be water soluble, or by direct microinjection of free drug (Figure 1A). Limitations of these passive delivery strategies include off-target effects and general toxicity that may preclude assessing any impact on macrophage function. Additionally, when investigating drug effects on macrophages it is unknown whether the drugs are acting on the macrophages themselves or through more indirect mechanisms. When performing similar chemical intervention studies to investigate macrophage function, we recognized there was an unmet need to develop an inexpensive and straightforward delivery method to target drugs specifically to macrophages.
Liposomes are microscopic, biocompatible, lipid bilayered vesicles that can encapsulate proteins, nucleotides and drug cargo19. The unilamellar or multilamellar lipid bilayer structure of liposomes forms an aqueous inner lumen where water-soluble drugs can be incorporated while hydrophobic drugs can be integrated into the lipid membranes. In addition, the physicochemical properties of liposomes, including size, charge and surface modifications can be manipulated to tailor their targeting to specific cells20,21. These features of liposomes have made them an attractive vehicle to deliver drugs and enhance the precision of current treatment regimens20. As liposomes are naturally phagocytozed by macrophages (a feature exploited by their routine use in delivering clodronate specifically to macrophages for ablation experiments22), they present as an attractive option for macrophage-specific drug delivery (Figure 1B).
This protocol describes the formulation of drugs into blue fluorescent liposomes coated with the hydrophilic polymer poloxamer 188, that forms a protective layer on the liposome surface and has been shown to enhance drug retention and have superior biocompatibility23. Poloxamer was chosen for surface coating of liposomes as our previous research had shown that, when compared to polyethylene glycol modified liposomes, poloxamer modified liposomes showed better biocompatibility following subcutaneous injection of rat paws and similar pharmacokinetics in rabbits following intravenous infusion23. Protocols are also described for their microinjection into larval zebrafish and live imaging to assess their macrophage-targeting ability and localization to intracellular compartments necessary for liposome degradation and cytoplasmic drug delivery. As a proof-of-concept we have previously used this technique to target two drugs to macrophages to suppress their activation in a larval zebrafish model of acute gouty inflammation24. This drug delivery technique expands the chemical &#34;toolkit&#34; available to zebrafish researchers wanting to ensure macrophage-targeting of their drugs of interest.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>850355P</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-diseteroyl-sn-glycero-3-phosphocholine (DSPE)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>850367P</td>
			<td></td>
		</tr>
		<tr>
			<td>1.0 &#181;m Whatman Nuclepore Track-Etched polycarbonate membranes</td>
			<td>GE Healthcare Life Sciences</td>
			<td>110610</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL round-bottom flask</td>
			<td>Sigma-Aldrich</td>
			<td>Z278262</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm culture dish</td>
			<td>Thermo Scientific</td>
			<td>150460</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34998</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1260 Infinity Diode Array Detector</td>
			<td>Agilent Technologies</td>
			<td>G4212B</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1260 Infinity Quaternary Pump</td>
			<td>Agilent Technologies</td>
			<td>G1311B</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1290 Infinity Series Thermostat</td>
			<td>Agilent Technologies</td>
			<td>G1330B</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti mini-extruder Avanti Polar Lipids Inc.</td>
			<td>Avanti Polar Lipids Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>borosilicate microinjection needles</td>
			<td>Warner Instruments</td>
			<td>203-776-0664</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C4901-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>cholesterol</td>
			<td>Sigma-Aldrich</td>
			<td>C8667</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont No.5 fine tip forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Microloader pipette tip</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf SmartBlock 1.5 mL, thermoblock for 24 reaction vessels</td>
			<td>Eppendorf</td>
			<td>4053-6038</td>
			<td></td>
		</tr>
		<tr>
			<td>eyelash manipulator</td>
			<td>Ted Pella Inc.</td>
			<td>113</td>
			<td></td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>Hawksley</td>
			<td>BS.748</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>BDH Chemicals</td>
			<td>441474J</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC system</td>
			<td>Agilent Technologies</td>
			<td>1260 series HPLC system</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>low melting point agarose</td>
			<td>Invitrogen</td>
			<td>16520-100</td>
			<td></td>
		</tr>
		<tr>
			<td>LysoTracker Deep Red</td>
			<td>Invitrogen</td>
			<td>L12492</td>
			<td>1 mM stock solution in DMSO, keep at -20 &#176;C and protect from light.</td>
		</tr>
		<tr>
			<td>LysoTracker Deep Red</td>
			<td>Thermo Scientific</td>
			<td>L12492</td>
			<td></td>
		</tr>
		<tr>
			<td>magnetic stand</td>
			<td>Narishige</td>
			<td>GJ-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Marina Blue 1,2-dihexadecanoyl-sn-glycero-phosphoethanolamine (Marina Blue DHPE)</td>
			<td>Invitrogen</td>
			<td>M12652</td>
			<td>Keep at -20 &#176;C and protect from light.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>methyl cellulose</td>
			<td>Sigma-Aldrich</td>
			<td>M0387-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>methylene blue</td>
			<td>Alfa Aesar</td>
			<td>42771</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>230391-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Narishige</td>
			<td>M-152</td>
			<td></td>
		</tr>
		<tr>
			<td>mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M-3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitochondria-targeting antioxidant MitoTEMPO</td>
			<td>Sigma-Aldrich</td>
			<td>SML0737</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoSOX Red Mitochondrial Superoxide Indicator</td>
			<td>Thermo Scientific</td>
			<td>M36008</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTEMPO</td>
			<td>Sigma-Aldrich</td>
			<td>SML0737</td>
			<td>Keep at -20 &#176;C and protect from light.</td>
		</tr>
		<tr>
			<td>N-Phenylthiourea (PTU)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629-10G</td>
			<td>Take care when handling, toxic.</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>BDH Chemicals</td>
			<td>27810.295</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (pH 7.4)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (100 mm x 20 mm)</td>
			<td>Corning Inc.</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenomenex C18 Gemini-NZ 3 mm 250 mm x 4.6 mm column</td>
			<td>Phenomenex</td>
			<td>00G-4439-E0</td>
			<td></td>
		</tr>
		<tr>
			<td>pHrodo Red Escherichia coli BioParticles Conjugate</td>
			<td>Thermo Scientific</td>
			<td>P35361</td>
			<td></td>
		</tr>
		<tr>
			<td>pHrodo Red Escherichia coli BioParticles Conjugate</td>
			<td>Invitrogen</td>
			<td>P35361</td>
			<td>Keep at -20 &#176;C and protect from light. Make 1 mg/mL stock solution by dissolving 2 mg lyophilized product in 2 mL of PBS supplemented with 20 mM HEPES, pH 7.4.</td>
		</tr>
		<tr>
			<td>plastic transfer pipette</td>
			<td>Medi&#39;Ray</td>
			<td>RL200C</td>
			<td></td>
		</tr>
		<tr>
			<td>poloxamer 188</td>
			<td>BASF Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pressure injector</td>
			<td>Applied Scientific Instruments</td>
			<td>MPPI-2</td>
			<td></td>
		</tr>
		<tr>
			<td>rotary evaporator</td>
			<td>B&#252;chi, Flawil, Switzerland</td>
			<td>B&#252;chi R-215 Rotavapor</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning confocal microscope</td>
			<td>Olympus</td>
			<td>Olympus FV1000 FluoView</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall WX+ Ultracentrifuge</td>
			<td>Thermo Scientific</td>
			<td>75000090</td>
			<td></td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td>Leica</td>
			<td>MZ12</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>A5040-25G</td>
			<td>Make 4 mg/mL stock solution (in deionzed H2O) and keep at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>triton-X100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic bath</td>
			<td>Thermo Scientific</td>
			<td>FB-11205</td>
			<td></td>
		</tr>
		<tr>
			<td>Volocity Image Analysis Software</td>
			<td>PerkinElmer</td>
			<td>version 6.3</td>
			<td></td>
		</tr>
		<tr>
			<td>water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zetasizer Nano</td>
			<td>Malvern Instruments Ltd</td>
			<td>Zetasizer Nano ZS ZEN 3600</td>
			<td></td>
		</tr>
	</tbody>
</table>

targeting drugs to larval zebrafish macrophages by injecting drug-loaded liposomes

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

The thin film hydration approach described here for the preparation of fluorescent liposomes enclosing drugs is a simple and cost-effective method. With the protocol used in this study, the liposomes are expected to be unilamellar23,24. The size, zeta potential, drug loading and entrapment efficiency of the liposomes produced are summarized in Table 1. The particle size of the liposomes (before and after drug loading) are similar (Table 1...

Watch this Scientific Journal Video about Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes at JoVE.com

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

This protocol describes a technique for targeting drugs of interest to larval zebrafish macrophages to manipulate and evaluate macrophage function. This technique has advantages over traditional drug delivery strategies such as immersion by ensuring that the drugs are specifically targeted to macrophages through liposome encapsulation. When applied to zebrafish models of disease with an inflammatory component, this technique has the potential to resolve how macrophages contribute to pathological processes.Here, we demonstrate the utility of this technique by targeting a mitochondrial-reactive oxygen species-inhibiting drug to macrophages to suppress activation. The microinjection of drug-loaded liposomes into larvae can be challenging, as care should be taken to not cause excessive epithelial damage, potentially influencing immunological studies. Visual demonstration of this protocol is important because it involves pharmacological and whole animal live cell methodologies that may not be routinely performed in a single laboratory.To prepare 10 milligrams of liposomes in a 25-milliliter round-bottom flask, add 31 nanomolars of marine blue and 300 nanomolars of a mitochondrial-reactive oxygen species-inhibiting drug to the flask. Sonicate briefly to fully dissolve, and remove the solvent slowly on a rotary evaporator set over a 45-degree Celsius water bath at 75 rotations per minute under vacuum pressure. When a dry lipid thin film has formed within the glass walls of the flask, further dry the film for 45 minutes under nitrogen to facilitate the complete removal of the organic solvent.Next, hydrate the film with one milliliter of PBS, and seal the flask with paraffin film before agitating the flask by hand over a 60-degree Celsius water bath for 20 minutes. After seven cycles of freezing and thawing, use a mini-extruder and one-micrometer track-etched polycarbonate membranes to extrude the liposomes for two to six cycles. When large unilamellar vesicles have been obtained, condense the fresh liposomes into a pellet by ultracentrifugation.Incubate the pellet with one milliliter of 1%poloxamer 188 solution for 30 minutes at 45 degrees Celsius and 750 rotations per minute in a ThermoMixer with a two-milliliter microcentrifuge tube attachment. During the post insertion of poloxamer 188 into the liposomes, the correct poloxamer concentration, incubation time, and temperature are important for achieving the optimal physicochemical characteristics of the resulting liposomes. At the end of the incubation, ultracentrifuge the mixture again under the same centrifuge conditions, and remove the supernatant containing any unbound polymer or drug.Then store the liposome pellets at four degrees Celsius, protected from light. To determine the size and zeta potential of the liposomes, dilute the liposome formulation with ultrapure water at a one-to-100 ratio. Use a dynamic light scattering analyzer to measure the particle size, polydispersity index, and zeta potential in triplicate at 25 degrees Celsius.To calculate the entrapment efficiency and drug loading of the liposomes, dissolve the liposomes in Triton X-100. Inject 20 microliters of the dissolved liposome sample into a high-pressure liquid chromatography system with a quaternary pump, a diode array detector set at 230 nanometers, and a three-micrometer, 250-times-4.6-millimeter column. To prepare transgenic zebrafish larvae for the injection, use fine-tip forceps to manually dechorionate 30 hours post-fertilization embryos, and allow the embryos to develop at 28.5 degrees Celsius until two days post-fertilization.When the embryos have reached the appropriate stage, dilute a freshly prepared liposome formulation at a one-to-one ratio in sterile PBS, and mix the solution for two to three seconds by vortexing. Pour enough 3%methyl cellulose in E3 medium into the lid of a small, 35-millimeter culture dish lid to form a thin film covering the entire surface. Use a plastic transfer pipette to collect a pool of approximately 20 to 25 anesthetized larvae.Allow the embryos to concentrate at the tip of the pipette by gravity, and carefully transfer the larvae into the middle of the 35-millimeter culture dish lid with minimal solution. Use a microcapillary loader to arrange the larvae with their anteriors toward the top of the injection plate with the dorsal surface facing toward the microinjection needle for injection into the hindbrain ventricle or with their anteriors toward the left of the injection plate with the ventral surfaces facing toward the microinjection needle for injection into the sinus venosus. Use a microcapillary loader to back-fill an injection needle with approximately five microliters of the liposome injection mix.Mount the needle into a micromanipulator connected to a magnetic stand and a pressure injector, and position the setup under a stereo microscope. Use clean, fine-tip forceps to cut the tip of the microinjection needle to generate an opening of approximately five micrometers in diameter. Set the injector to a pulse duration of approximately 50 milliseconds and an injection pressure of approximately 40 pounds per square inch.Inject a bolus into a drop of mineral oil positioned over the grid lines of a hemocytometer to calibrate the volume to be injected. Then adjust the pulse duration and/or the injection pressure until the diameter of the bolus covers 2 1/2 of the smallest grid lines. Carefully inject one nanoliter of the liposome injection mix into the hindbrain ventricle of each larvae.Immediately following the injections, add E3 medium to the injection plate, and use a plastic transfer pipette to gently agitate the larvae out of the methyl cellulose. To image the dorsal surface of the hindbrain on a confocal microscope equipped with a water immersion lens, first fill a 35-millimeter culture dish to a depth of five millimeters with mounting medium, and let the medium polymerize. Excavate a small trench in the agarose of sufficient area to accommodate the yolk sac of the experimental embryo, and use a plastic transfer pipette filled with molten mounting medium to collect an anesthetized larva.Immediately deposit the larva into the culture dish, and use a microcapillary loader to orientate the yolk sac ventral side down into the excavated holes. Maintain the embryo in this position until the agarose polymerizes. Overlay the embryo with E3 medium supplemented with 200 micrograms per milliliter of tricaine.To live image the hindbrain ventricle, select the 20x objective, and set the confocal microscope to 512 by 512 pixels and a 2.5x zoom. When the microscope parameters have been set, acquire 40-by-three-micrometer Z-stacks extending from dorsal-most surface of the hindbrain using two-channel imaging to image the blue fluorescent liposomes and green fluorescent macrophages. When all of the images have been obtained, load the files into an appropriate 3D image analysis software program.Under the Measurement tab, drag the Find Objects protocol into the Drag Tasks Here To Make Measurements space. Select the DAPI channel to highlight the liposomes, and drag the Inspect tool through the Z-sections to detect the liposome-laden macrophages. To quantify the fluorescence intensity of liposomes within the individual cells, drag the Clip To Regions Of Interest protocol into the Drag Tasks Here To Make Measurements space, and use the Rectangle tool to draw a box around the cell of interest in the X, Y, and Z dimensions.Then select Make Measurement Item from the Measurements dropdown menu to generate a measurement file in the library window. In this table, the size, zeta potential, drug loading, and entrapment efficiency of the produced liposomes are summarized. As observed, the particle size of the liposomes is similar with and without drug loading, although the surface charge of drug-loaded liposomes is slightly more neutral than the control liposomes with marine blue only.Microinjection of marine blue-labeled liposomes into the hindbrain ventricle, as demonstrated, results in a rapid uptake by resident macrophages that can be readily quantified by confocal microscopy by three hours post-injection. Neutrophils, however, are rarely observed to contain intracellular liposomes. Within individual liposome-laden macrophages, the liposomes accumulate within the phagolysosomal compartments, which is necessary for liposome degradation and subsequent drug content release into the cytoplasm.Delivery into the sinus venosus can deliver the liposomes to caudal hematopoietic tissue-resident macrophages via the circulation. Microinjection of liposomes loaded with the mitochondrial-reactive oxygen species-inhibiting drug into the hindbrain ventricle can significantly suppress monosodium urate crystal-driven mitochondrial-reactive oxygen species production within liposome-laden hindbrain-resident macrophages. Further validation of the suppressive effects of drugs of interest on macrophage activation states can be performed by investigating proinflammatory cytokine gene expression by whole mount in situ hybridization and the temporal recruitment of neutrophils.Using this technique, we've been able to target drugs to macrophages to confirm that an immunometabolic mechanism drives their activation in a larval zebrafish model of acute gouty inflammation. Future adaptions of this protocol could include performing surface modification of the liposome to enhance macrophage targeting or to promote targeting to other immune cells, such as neutrophils.

targeting-drugs-to-larval-zebrafish-macrophages-injecting-drug-loaded

Research

JoVE Journal

Immunology and Infection

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

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 ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...