Name: visualization of il-22-expressing lymphocytes using reporter mice
Uploaded: 2017-01-25T12:30:00
Description: Watch this Scientific Journal Video about Visualization of IL-22-expressing Lymphocytes Using Reporter Mice at JoVE.com

We thank Kelli Czarra and Megan Karwan for animal technical assistance, Kathleen Noer and Roberta Matthai for flow cytometry assistance, and Donna Butcher andMiriam R. Anver for pathology analysis. This project was supported by a grant from the Ely and Edythe Broad Foundation (to Scott Durum) and has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E (MRA).

All animals received proper care in accordance with the experimental procedures outlined in the 2011 Guide for Care and Use of Laboratory Animals Committee of Frederick National Laboratory for Cancer Research.
1. Generation of IL-22-tdTomato Reporter Mice by BAC Recombineering
NOTE: The mice should be unconscious and do not move in response to a noxious stimulus. Sterilize the surgical area with 70% ethanol and sterilize all surgical tools using a glass bead sterilize...

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IL-22 plays an essential role in innate host defense and tissue remodeling. IL-22-producing cells have been identified ex vivo by intracellular staining. However, it still remains difficult to track IL-22 expression in situ, either in the normal state or in inflammatory conditions. This protocol describes a novel method to develop an IL-22 reporter mouse model, which enables us to localize the reporter-expressing cells in vivo. The reporter gene encoding TdTomato was inserted into the IL-22 loc...

Cell type-specific expression of reporter genes is useful to identify cells actively expressing the target in tissues under homeostatic and perturbed states. It also allows for the purification of these cells, which remain viable, to study their other properties. Reporter mice have been utilized in elucidating the mechanism of action for specific cytokines, transcription factors, and regulatory elements. Previous strategies1,2,3 have largely relied on knocking the reporter into the target locus in the mouse chromosome, a time-consuming and costly procedure. Thus, a simpler method for the generation of reporter mice is desirable.
Cytokines are a broad class of small, secreted proteins/peptides that regulate immune responses through intercellular signaling. Interleukin 22 (IL-22) is a cytokine with many reported activities, including barrier function, tissue repair, and inflammation4. Although IL-22 was initially discovered as a T-cell product5, subsequent reports demonstrated its expression in natural killer (NK) cells in humans6 and mice7 and in other classes of innate lymphocytes8. Despite extensive observation of IL-22-producing cells, visualization of IL-22 previously required ex vivo stimulation and permeabilization to stains with antibodies. Therefore, novel IL-22 reporter mice would be a very useful tool to investigate the function of IL-22 in homeostatic and pathogenic processes.
Here, we developed a simplified transgenic reporter mouse model to observe the IL-22-producing cells in vivo and in vitro. Using a BAC recombineering method9, we inserted the tdTomato cDNA sequence with Poly A signal fragments into the IL-22 locus and replaced exon 1. The other untranslated regions, exons, and regulatory elements were not perturbed, since we would like to mimic the natural regulation of IL-22 as much as possible. The site of reporter insertion disrupts the signal sequence, resulting in the accumulation of the reporter inside the producing cells, unlike IL-22 itself, which is rapidly secreted. This new method can also be applied to the generation of reporter mice for other secreted proteins.

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visualization of il-22-expressing lymphocytes using reporter mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

A murine IL-22 reporter transgene was created using recombineering to modify a bacterial artificial chromosome carrying the IL-22 locus. Figure 1 shows a diagram of pBACe3.6 vector containing the sacBII gene, a positive-selection marker, and chloramphenicol antibiotic resistance gene11. After introducing tdTomato into exon 1, the signal peptide sequence was disrupted, as shown in Figure 2. Thus, the tdTomato reporter was t...

Watch this Scientific Journal Video about Visualization of IL-22-expressing Lymphocytes Using Reporter Mice at JoVE.com

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

The overall goal of this mouse genetic modification procedure is to identify, purify, and characterize IL-22-producing immune cells. Cells of the immune system release mediators called cytokines which are responsible for defending us against foreign agents like bacteria and restoring damaged tissues. IL-22 is one such cytokine and has been ascribed many activities ranging from tissue repair to pathology.And a number of different cell types in the immune system have been reported to be capable of producing it. So to clarify which cells actually produce IL-22, we developed a new IL-22 reporter mouse. This enabled us to identify relevant IL-22 producers in vivo in mice under normal conditions and in an inflammatory bowel disease model.We also used reporter cells to purify IL-22 expressors to determine their other properties. The method of generating reporter mice is briefly outlined and the methods of analysis are described. The methods described can be applied to other genes for purposes of identifying the expressors and purifying viable expressing cells for further characterization.This method can help answer key questions in immunology field about the biological activities of IL-22 and the properties of the cells that produce it. Demonstrating the procedure with me will be Julie Hixon, a biologist, Caroline Andrews, a post doc, and Wenqing Li, a staff scientist all from Dr.Durham's lab. After inserting the tdTomato reporter gene into a shuttle vector, replacing the GFP gene within the same site, use the appropriate bacterial artificial chromosome, or BAC template to amplify the A and B homology boxes to the first translated exon of IL-22 in a 50 microliter PCR reaction system.Then transform the purified vectors into BAC competent bacterial cells on Luria-Bertani or LB agar plates containing chloramphenicol and ampicillin at 37 degrees Celsius. At the end of the incubation, inoculate two to three colonies in one milliliter of LB broth, supplemented with chloramphenicol at 37 degrees Celsius and 225 rpm. After one hour, spread 100 microliters of each mixture onto an LB agar plate supplemented with chloramphenicol and sucrose for an overnight culture at 37 degrees Celsius.The next morning, replate the big colonies onto a new LB agar plate with chloramphenicol and the small colonies onto another LB agar plate and expose the plates to UV light for 30 seconds. Then select a UV sensitive colony and use a tdTomato IL-22 heterozygous primer to confirm the presence of the modified BAC DNA by PCR. After removing the spleen and thymus, use fine point serrated dissection forceps to harvest the pearly white mesenteric lymph nodes close to the wall of the small intestine.Next, remove the entire small intestine and colon and use scissors to extract the extended oval Peyer's patches along the gut. When all of the lymph tissues have been collected grind the mesenteric lymph nodes and Peyer's patches together between two frosted slides in PBS supplemented with FBS on ice. After generating individual spleen and thymus single cell suspensions in the same manner, centrifuge the spleen cells followed splenic Red Blood Cell Lysis with one milliliter of ACK Lysing Buffers spleen.After one minute, restore the isotonicity with nine milliliters of PBS and centrifuge the cells again. Resuspend the pellet in five milliliters of PBS and filter the cells through a 100 micron cell strainer. Then collect the cells by another centrifugation, resuspending the pellet in five milliliters of RPI medium for enumeration by trypan blue exclusion.After harvesting the small intestine from the duodenum to the ileum along with the entire colon use forceps to remove the extra adipose and mesenteric tissue from the intestine and immediately place the bowel tissue in ice cold PBS. Use forceps to harvest the Peyer's patches along the distal region of the small intestine. Then use scissors to open the intestine lengthwise and thoroughly flush the tissue with 10 milliliters ice cold PBS.Transfer the sample into 20 milliliters of predigestion buffer for two 30 minute incubations at 37 degrees Celsius and 50 rpm in a shaker vortexing the tissue for 30 seconds at 12, 000 rpm and pressing all of the fragments through a 100 micron cell strainer at the end of each incubation to collect the epithelial cell layer. Next, add 20 milliliters fresh EDTA solution to the cell slurry for another 30 minute incubation with shaking at 37 degrees Celsius, filtering and cooling the intestinal epithelial cells at the end of the incubation as just demonstrated. Place the remaining intestine fragments on ice and collect the pooled intestinal epithelial cells by centrifugation, followed by a wash in 10 milliliters of HBSS supplemented with FBS.Resuspend the washed pellet in eight milliliters of 44%colloidal silica medium and overlay the cell suspension with five milliliters of 67%colloidal silica medium. Separate the cells by centrifugation and use a one milliliter pipette to remove the top five milliliter of cell separation medium. Then collect the cells at the interface and wash the intestinal epithelial cells in 10 milliliters of RPMI medium, resuspending the pellet in five milliliters of fresh RPMI.After preparing single spleen cell suspensions from IL-22 td-Tomato mice as just demonstrated. Resuspend the cells at a one times 10 to the seventh cells per milliliter concentration in sterile PBS and use the mouse CD4 cell negative isolation method to purify the CD4 positive T cells. Next, label the T cells with anti-CD4 and anti-CD45RB antibodies for 30 minutes at four degrees Celsius.At the end of the incubation, collect the cells by centrifugation followed by a wash in two milliliters of PBS supplemented with FBS. Resuspend the washed pellet in one milliliter of fresh PBS and FBS and sort the labeled T cells by flow cytometry by their CD4 and CD45RB coexpression. When all of the cells have been sorted, collect them by centrifugation and resuspend the pellet at a one times 10 to the six cells per milliliter concentration in sterile PBS for injection of the cells into RAG1 knockout mouse recipients.To create the murine IL-22 reporter transgene recombineering was used to modify a bacterial artificial chromosome carrying the IL-22 locust that also contains a positive selection marker and a chloramphenicol antibiotic resistance gene. After introducing tdTomato into exon1, the signal peptide sequence was disrupted, trapping the tdTomato reporter inside the IL-22 expressing cells and enabling their detection and isolation by flow cytometry. To test the fidelity of the IL-22 reporters, in vitro generated splenocytes can be cultured under Th22 or neutral conditions with the IL-22 tdTomato expression apparent by both flow cytometry and fluorescent microscopy.In vivo, the IL-22 reporters detected in different mouse tissues under homeostatic conditions with most of the signal observed in the lamina propria cells from the gut, but not in the axillary lymph node, spleen or thymus. In a mouse colitis model induced by the transfer of reporter CD4 positive CD45RB high T cells into RAG1 knockout mice, the tdTomato reporters are first visualized in the mesenteric lymph nodes, but their eventual accumulation with in the lamina propria of the distal small intestine and colon tissues. While attempting this procedure it's important to remember to choose the right BAC clone.Following this procedure, other methods, like the establishment of lung or skin infection models can be performed using this reporter mouse to answer additional questions about the role IL-22 in host defense and tissue repair. After it's development this technique paved the way for researchers in the field of immunology to explore the function of secreted proteins that demonstrate a low expression using a mouse model such as this. After watching this video, you should have a good understanding of how to generate a transgenic reporter mouse and to identify IL-22 reporter genes in vitro and in vivo.

visualization-of-il-22-expressing-lymphocytes-using-reporter-mice

Research

JoVE Journal

Immunology and Infection

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal &#8212; used as a measure of priming 2) in vivo neutrophil labeling &#8212; achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models.

Intravital Imaging of Neutrophil Priming Using IL-1&amp;#946; Promoter-driven DsRed Reporter Mice

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...