Name: facilitating drug discovery: an automated high-content inflammation assay in zebrafish
Uploaded: 2012-07-16T13:00:00
Duration: 7 min 50 s
Description: Watch this Scientific Journal Video about Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish at JoVE.com

We thank Nadeshda Wolf, Nadine Eschen and Susanne Delong for excellent animal husbandry. CW was supported by a PhD fellowship from the Helmholtz program 'BioInterfaces international graduate school' (BIFIGS). Further support was provided by a KIT-RISC grant and by a Marie Curie International Reintegration Grant within the 7th European Community Framework Program (PIRG07-GA-2010-267552) to CG. AHS was supported by BOLD (Biology of Liver and Pancreatic Development and Disease) - Marie Curie Initial Training Network (238821). UL was supported by Dopaminet FP7 (Seventh Framework Program) (223744).

1. Animal Handling
For the ChIn assay use 3 dpf larvae arising from group matings between homozygous double transgenic cldnB::GFP/lysC::DsRED2 and AB (wild-type) fish. Collect embryos by natural spawning and raise them at 29 &deg;C in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 0.1% methylene blue, equilibrated to pH 7.0) in Petri dishes. It is essential to maintain a density of embryos not exceeding 50-70 per plate.
2. Larva Sorting
Check all larvae under a fluorescence stereoscope for fluorescent reporter expression, spontaneous inflammation and appropriate age related development.
3. Screening Medium Preparation (Always prepare fresh)
Prepare E3 medium without methylene blue supplemented with DMSO (1% final) and MS222 (0.05 g/l).
4. CuSO4 Preparation (Always prepare fresh)
First weigh out CuSO4 (Mr=159.6 g/mol) and prepare a 20 mM stock solution in dH2O. Prepare 120 &mu;M CuSO4 working solution (from 20 mM stock solution) in E3/DMSO(1%)/MS-222. Protect CuSO4 solution from light.
5. 384-well Plate Preparation (Greiner 384 Well Microplate)
Pre-add 20 &mu;l of E3/DMSO(1%)/MS-222 to each well with a reverse pipette. An increased pipette tip bore is required to handle the embryos carefully without inflicting any wounding; this is done by cutting the tip to a 2 mm bore. Transfer single larva in 74 &mu;l of medium to each well (84 &mu;l for untreated control). If necessary, orient larvae in a lateral position within the well using a flexible Eppendorf Microloader Pipette Tip (Eppendorf; 5242 956.003).
Conduct all following liquid handling steps with a robot liquid handling workstation to ensure simultaneous treatment of all larvae.
6. Drug Treatment
Mix compounds in drug stock plate 5 times by pipetting up and down. Add 16 &mu;l of 7.5X drug stock plate to each well and mix 5 times. Adjust tip position within wells to prevent injury of larvae and dispense the medium at 10 &mu;l/s. Mix medium in wells 4 times to ensure homogenous distribution of the drug within the well.
7. Incubation
Incubate screening plate for 1 h at 29 &deg;C covered with aluminum foil to protect compounds as well as CuSO4 from light.
8. Chemical Wounding
Add 10 &mu;l of 120 &mu;M CuSO4 working solution to each well except to negative controls, mix 4 times and incubate again for 1 h at 29 &deg;C.
9. Washing
Remove and exchange 80 &mu;l of medium from each well twice (in 20 &mu;l steps) to remove compounds and CuSO4.
10. Image Acquisition
Start image acquisition on an inverted automated microscope (i.e.: Olympus scan^R) 90 minutes after initial copper treatment. Set initial z-level so that neuromasts from right and left posterior lateral line are visible. Image each well once per hour in the channels brightfield, Cy3 and GFP in 4 focal planes (50 &mu;m distance) using a 4x objective (N.A. = 0.13).
Additional information on image and data processing are available upon request.
11. Image Processing
1. Data sorting
Raw images are processed with our custom LabView software script. The first operation in the image processing pipeline is sorting of raw images from the microscope generated data folder by channel, well and time-point information.
2. Extended focus
Subsequently the software creates extended focus images from 4 focal planes for each of the channels.
3. RGB-overlay
In a last step the extended focus images from the 3 channels are merged to result in a final RGB-overlay image.
4. Automated neuromast detection
A pattern recognition tool (LabView Rapid IA prototyping tool) identifies neuromasts within the RGB overlay images and creates an empirically defined area of interest around the neuromasts.
5. Quantification
Within the empirically defined area of interest surrounding injured neuromasts red fluorescent leukocytes (reflected as red pixels) are scored resulting in a primary readout of percent area occupied by leukocytes (Paol). On average 95.27 &plusmn; 2.11% (FDA1) and 95.12 &plusmn; 1.56% (FDA2) of wells (larvae) have been detected properly and have subsequently been subjected to further data processing. The raw data output (Paol for each detected neuromast) is stored in a txt file and serves as the data input for the MATLAB scripts that process the raw data further.
12. Data Processing
1. iMaps
Assessing the success of individual experiments by graphical visualization of the raw data output Paol in a color code could be realized with so-called inflammation maps (iMaps) (Figure 2). Bright green reflects a high initial inflammatory index; black indicates no inflammation. This quick overview allows for rapid identification of failed experiments, which can then be excluded from further data analyses.
2. Averaging of controls
Each experimental plate contains 320 compounds reflecting a single data point per compound and time-point as well as 32 positive and negative controls, respectively. The 32 control replicates are averaged and standard deviation is calculated. For the controls only data points within 2 standard deviations are included.
3. Normalization
Normalization is done by spreadsheet analysis. The average value of DMSO, being the negative control, is set to 0 and the highest averaged Paol for the copper control is set to 1, so that the maximum difference between the positive and negative control is set to 1. Each compound's Paol is linearly interpolated or extrapolated to the respective controls on the experimental plate.
4. Final read-out: Inflammatory index
After sufficient replicate experiments (i.e. 15) have been conducted, normalized raw data from replicate experiments are averaged, resulting in a final read-out - the inflammatory index. The initial inflammatory index of the copper control starts at 100% for time-point zero and correlates to image acquisition start time (90 minutes after initial copper treatment).
5. Monotonic exponential regression fitting
Due to inflammation resolution over time we perform a monotonic exponential nonlinear regression fitting towards the initial inflammation by using <img src="/files/ftp_upload/4203/4203eq1.jpg" alt="Equation 1" />- a0 is a measure for the initial response at t=0, a1 is related to the slope of the magnitude over time.
6. 2-D feature space plot (Figure 4)
To generate the feature space, a non-linear regression is applied and a cluster-analysis divides the feature space into characteristic regions, thus allowing automated identification of interesting candidates from different immune-modulatory categories (anti-inflammatory, anti-resolution, pro-inflammatory, pro-resolution). All compounds are displayed in the 2-D plot based on the parameters a0 and a1.
13. Data Handling and Storage
Our data handling routines create webpages to visualize and represent such drug screens providing a quick overview of the image and data processing steps' results, as well as a detailed view when requested by the user10. Soft links to other relevant heterogeneous biological and chemical databases are integrated as well to provide a valuable resource for comparative and novel studies11. By these routines, proper data standards and metainformation are set for long term storage of this data.
14. Representative Results
In a pilot screen we analyzed a library consisting of 640 FDA approved known bioactive compounds for effects on initiation, progression or resolution of a granulocytic inflammatory response. Based on the observed effects we classified 4 types of immune modulatory phenotypes that may be indicative of different modes of action: anti-inflammatory (1), anti-resolution (2), pro-inflammatory (3) and pro-resolution (4), which can be described with the parameters a0 and a1, that arise from a monotonic non-linear regression fitting (eao+alx-)towards the initial inflammation. A0 represents the magnitude of the inflammatory response whereas a1 characterizes the slope of the curve and thus describes inflammation resolution. Displaying all compounds in a 2-D feature space plot (a1 vs. a0) allows for automated identification of hit candidates from the different immune modulatory categories (Figure 3).
45 out of 640 compounds exerted significant anti-inflammatory effect by reducing the initial inflammatory index to 50% or less. Within this category we found 6 compounds belonging to the pharmacological class of non-steroidal anti-inflammatory drugs (NSAIDs), confirming the validity of our approach. However, NSAIDs ranked not amongst the most potent anti-inflammatory drugs. Apart from the NSAIDs we found several additional pharmacological drug classes such as for example Angiotensin receptor blockers (ARBs), antibiotics and proton pump inhibitors.
7 compounds met the empirically defined threshold criteria for potential pro-resolution drugs.
18 drugs exerted a pro-inflammatory effect, resulting in exaggerated leukocyte recruitment to injured neuromasts compared to the positive controls.
Only 2 compounds prevented timely resolution of the inflammatory response.
The anti-inflammatory effect of several hit candidates (exemplary candidates from above mentioned pharmacological drug classes) from the pilot screen could be confirmed in a dose-dependent manner in subsequent secondary ChIn assays (data not shown). Retesting of 4 potential pro-resolution drugs did not confirm the indicated mode of action as exerted in the primary screen. 2 of the drugs had a slight anti-inflammatory potential at higher concentrations (20 &mu;M). A third drug exhibited a marginal non-dose-dependent anti-inflammatory effect at drug concentrations ranging from 5 - 20 &mu;M. The fourth drug had no effect on the inflammatory response at the concentrations tested.
<img alt="Figure 1" src="/files/ftp_upload/4203/4203fig1.jpg" /> 
Figure 1. Workflow of the ChIn assay. Individual compound transgenic cldnB::GFP/lysC::DsRED2 larvae (3 dpf) are manually distributed in 384-well microtiter plates. Drugs are added simultaneously to each well using a Zephyr Compact Liquid Handling Workstation. Assay plates are then incubated for 1 h at 29 &deg;C. Treatment with CuSO4- solution (10 &mu;M final concentration) for 1 h inflicts the wounding and initiates an acute inflammatory response. Finally, copper solution and drugs are washed off and automated image acquisition is started (90 minutes after initial copper treatment).
<img alt="Figure 2" src="/files/ftp_upload/4203/4203fig2.jpg" /> 
Figure 2. Overview picture of raw images. The figure shows raw images of 4 focal planes (Z=0 - 3) at distances of 50 &mu;m in the channels Cy3 (left) and GFP (right), respectively. Arrows (Z=1,2) indicate exemplary neuromasts, arrowheads (Z=1,2) point to clustered leukocytes around neuromasts.
<img alt="Figure 3" src="/files/ftp_upload/4203/4203fig3.jpg" /> 
Figure 3. Inflammation Maps (iMaps). The success of individual experiments can be assessed in iMaps reflecting the inflammatory response (raw data) in a color code: Bright green reflects a high initial inflammatory response; black indicates no inflammation. iMaps at timepoint 0 (left) and time-point 5 (right) clearly show whether an experiment should be included in further data processing. Copper controls (row 13 and 24) show up in varying shades of green, whereas DMSO control wells (row 1 and 12) appear in dark green or black. At time-point 5, inflammation is almost resolved in copper controls, now indicated in dark shades of green or black. <a href="https://www.jove.com/files/ftp_upload/4203/4203fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/4203/4203fig4.jpg" /> 
Figure 4. 2-D feature space plot of 320 compounds of an FDA approved library and their respective controls. All compound wells can be displayed in a 2-D feature space plot based on the parameters a0 and a1, which arise from a non-linear regression fitting ( <img src="/files/ftp_upload/4203/4203eq1.jpg" alt="Equation 1" /> ) towards the initial inflammation. This feature space plot allows automated identification of interesting candidates belonging to one of the following 4 immune-modulatory categories that may be indicative of the drugs' mode of action: anti-inflammatory (1), anti-resolution (2), pro-inflammatory (3) and pro-resolution (4). <a href="https://www.jove.com/files/ftp_upload/4203/4203fig4large.jpg" target="_blank"> Click here to view larger figure</a>.

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No conflicts of interest declared.

Here we present a fully automated high-throughput screening strategy enabling identification of immune-modulatory compounds in a whole animal context. Thus allowing monitoring of an acute inflammatory response in vivo and in real time.
 We provide a protocol to apply zebrafish as a valuable model organism for drug discovery or drug repurposing efforts, since drug effects on leukocyte recruitment to wounds can be studied with relative ease in the context of a fully functional innate immune system. The unique advantage of our approach is the fact that chemical wounding renders manual larvae manipulation obsolete, thus permitting automation and screening in a high-throughput format.
 Additionally, adverse or toxic drug effects as well as strong off-target effects can be easily assessed with the proposed set-up. The FDA approved library however did not contain any toxic compounds at the given screening concentrations. Nevertheless, we propose for apparent toxic compounds to compare the available dataset (e.g. immune modulatory phenotype, drug concentration) between members of the same pharmacological substance class present in the tested library. Toxic members of a substance family, that otherwise exerts a promising immune modulatory phenotype, could be retested at lower concentrations, if need be.
 We have demonstrated the possibility of categorizing drugs automatically according to their exerted immune-modulatory effect. Drugs belonging to the category of anti-inflammatory compounds could be validated in independent experiments, most of the compounds exerting their effect in a dose-dependent manner. Four compounds identified as potential pro-resolution drugs could thus far not be confirmed as such. It also indicates that this particular immune modulatory category is difficult to describe, since no drugs solely affecting resolution have so far been described in literature. However, 3 of the 4 retested drugs showed an anti-inflammatory trend at higher molar concentrations. Optimizing the empirically defined threshold criteria should help to reduce the number of false-positives.
 Furthermore, the presented technique offers the possibility to be utilized for the identification of mutations in genes affecting leukocyte migratory behavior8.
 Concluding, the proposed method can facilitate and accelerate drug discovery or drug repurposing efforts by yielding potential new lead structures with readily available information on drug tolerance, toxicity, dosage as well as potential off-target effects. Therefore, serving as a simple, cost- and time-saving strategy at early stages of drug development.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Dimethyl sulphoxide</td>
 <td>Carl Roth GmbH &amp; Co KG</td>
 <td>A994.2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Copper(II) sulphate anhydrous</td>
 <td>Carl Roth GmbH &amp; Co KG</td>
 <td>PO23.1 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FDA approved drug library</td>
 <td>Enzo Life Sciences</td>
 <td>BML-2841-0100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>384 microwell plate Non-binding, &mu;Clear</td>
 <td>Greiner</td>
 <td>781906 </td>
 <td>black</td>
 </tr>
 <tr>
 <td>Olympus scan^R</td>
 <td>Olympus</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Zephyr Compact Liquid Handling Workstation</td>
 <td>Caliper</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
 </table>

facilitating drug discovery: an automated high-content inflammation assay in zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Watch this Scientific Journal Video about Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish at JoVE.com

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

facilitating-drug-discovery-an-automated-high-content-inflammation

Research

JoVE Journal

Immunology and Infection

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...