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 sterilizer.
<ol>
	<li>Purify BAC DNA (RP23-401E11, clone length 228391 bps) using an alkaline extraction method10,11. Characterize the BAC clone by SpeI digestion of 5 &#181;g of RP23-401E11 to confirm the correct size of the target gene. NOTE: The BAC DNA was digested into 14 fragments.</li>
	<li>Insert the TdTomato reporter gene into the Not I/Sal I site of the PLD53SC-A-GFP-B shuttle vector11 and replace the GFP gene in the same site. Then, using BAC RP23-401E11 as a template, amplify the homology boxes (A and B) to the first translated exon of Il-22 (exon 1) by PCR (95 &#176;C 2 min; 30 cycles of 95 &#176;C for 30 sec, 55 &#176;C for 30 sec, and 72&#176;C for 30 sec; and a final extension at 72 &#176;C for 10 min). In a 50 &#181;l PCR reaction system, add 50 ng (1 &#181;l) of BAC DNA, 0.5 &#181;l of primers (10 &#181;M), 40 &#181;l of PCR superMix high fidelity, and 8 &#181;l of H2O. 
	NOTE: The primers for the A and B boxes are as follows: A box Forward: 5&#39;- TGGCGCGCCGGAGCTGTGAAGAAAG and Reverse: 5&#39;- CAGAGATCGCACAAGTGTCAAC; B box Forward: 5&#39;- GTTAATTAACTGCCCGTCAACA and Reverse: 5&#39;- TGGCCGGCCTGAGCACCTGCTT CATC10. The sites of the restriction enzymes (Asc I and Pac I) are underlined.</li>
	<li>Ligate 500 ng of Asc I-digested A and 500 ng of Pac I-digested B fragments into 50 ng of PLD53SC-ATB (tdTomato) vector at 16 &#176;C for 1 hr.</li>
	<li>Transform the purified PLD53SC-ATB vector into BAC-competent cells11 and culture them on Luria-Bertani(LB) agar plates with chloramphenicol (20 &#181;g/ml) and ampicillin (30 &#181;g/ml) at 37 &#176;C. Pick two to three colonies of co-integrate from the Ch/Amp master plates.</li>
	<li>Inoculate each colony into 1 ml of LB supplemented with 20 &#181;g/ml chloramphenicol and incubate them for 1 hr at 37 &#176;C and 225 rpm. Spread 100 &#181;l of each mixture on a LB-agar plate (10 g of Peptone 140, 5 g of yeast extract, 12 g of agar, and 1 L of water; pH 7.5) supplemented with chloramphenicol and 4-4.5% of sucrose. Incubate the culture overnight at 37 &#176;C.</li>
	<li>Pick both big and small colonies and replate them on two LB-agar plates with chloramphenicol. Then, expose the plates either with or without UV light for 30 sec and pick the UV-sensitive colonies for further analysis.</li>
	<li>Identify the modified BAC DNA by PCR using tdTomato/IL-22 heterozygous primer (Forward: 5&#39; ACT TgT gCg ATC TCT gAT ggT; Reverse: 5&#39; TgT AAT Cgg ggA TgT Cgg C). The thermocycler conditions are as follows: 95 &#176;C for 2 min; 30 cycles of 95 &#176;C for 30 sec, 56 &#176;C for 30 sec, and 72 &#176;C for 30 sec; and a final extension at 72 &#176;C for 10 min.</li>
	<li>Confirm the sequence of the modified area of the BAC by direct BAC sequencing of large-prep BAC DNA12.</li>
	<li>Anesthetize a recipient mouse with a dose of 0.25% tribromoethanol into the peritoneal cavity when implanting the microinjected zygotes into pseudo-pregnant C57BL/6c mice.</li>
	<li>Linearize the IL-22-tdTomato BAC construct (10 &#181;g) by digestion with 5 &#181;l of restriction endonucleasePi-SceI in 100 &#181;l of the reaction system and microinject the linearized BAC DNA into fertilized eggs of C57BL/6 females at the pronuclear stage13. Screen the transgenic mice by Southern blot analysis of DNA isolated from tail biopsies using standard procedures.</li>
</ol>
2. Single-cell Preparation from Spleens, Thymus, Lymph Nodes, and Peyer&#39;s Patch
NOTE: IL-22-tdTomato reporter mice were maintained at the National Cancer Institute (NCI, Frederick, MD). The euthanasia method conforms to the most recent AVMA Guidelines on Euthanasia. All mice were euthanized using CO2 inhalation14.
<ol>
	<li>Euthanize an IL-22-tdTomato mouse in a CO2 chamber and put it on a clean pad. Sterilize the abdomen skin by spraying 70% alcohol and cut it open. Remove the spleen in the left flank and the thymus behind the sternum.</li>
	<li>To harvest the mesentericlymph nodes (located in the mesenteric tissue) and Peyer&#39;s patches (small lymphoidnodules throughout the ileum region of the small intestine), take the pearly white mesenteric nodes close to the wall of the small intestine using dissect forceps with fine-point serrated tips first and then remove the whole small intestine and colon out of the body. Find the extended oval patches (Peyer&#39;s patches)along the gut and cut them with scissors. Grind these lymphoid tissues (lymph nodes and Peyer&#39;s patches) individually between two frosted slides into a single-cell suspension in PBS/1% fetal bovine serum (FBS) buffer on ice.</li>
	<li>Mince the spleen or thymus tissues using the same method as in step 2.2. Centrifuge the splenic suspension for 8 min at 500 x g and 4 &#176;C. Add 1 ml of ACK lysing buffer per spleen to the cell pellets.</li>
	<li>Mix well and let them stand for 1 min. Add 9 ml of sterile phosphate-buffered saline (PBS) buffer to each sample. Spin down by centrifugation for 8 min at 500 x g and 4 &#176;C to remove the red blood cells. 
	NOTE: Do not add ACK lysing buffer to the thymus cells.</li>
	<li>Resuspend the resulting leukocytes in 5 ml of PBS and pass the cells through a 100 &#181;m cell strainer. Collect the cell pellets by centrifugation at 500 x g for 8 min.</li>
	<li>Resuspend the cells in 5 ml of RPMI media or FACS buffer and count the viable cells using 0.4% trypan blue dye/PBS solution.</li>
</ol>
3. Isolation of Intraepithelial Lymphocytes and Lamina Propria Cells from the Intestines
<ol>
	<li>Open the abdominal skin as in step 2.1, cut the small intestine, from duodenum (next to the stomach) to ileum (close to the appendix), and the whole colon, right above the anus. Remove the extra adipose and mesenteric tissue using forceps. Put the intestines immediately in ice-cold PBS.</li>
	<li>Look for Peyer&#39;s patches (small, rough nodules) along the distal region of the small intestine. Remove the masses using forceps andopen the intestine lengthwise using scissors. Thoroughly flush the intestine with 10 ml of ice-cold PBS.</li>
	<li>Incubate the tissues in 20 ml of pre-digestion buffer (5 mM EDTA and 1 mM dithiothreitol in Hank&#39;s Balanced Salt Solution (HBSS)) for 30 min at 37 &#176;C with slow rotation (50 rpm) in a shaker. After each of 2 incubations, remove the epithelial cell layer, containing the intraepithelial lymphocytes (IELs), by intense vortexing (30 sec at 12,000 rpm) and pass all the tissues through a 100 &#181;m cell strainer.</li>
	<li>Add 20 ml of new EDTA solution to the tissue for second incubation for 30 min at 37 &#176;C. Pass the IEL fractions through a 100 &#181;m cell strainer and pool them with the previous fractions. Keep the remaining intestine fragments on ice to be treated in step 3.9.</li>
	<li>Centrifuge the pooled IEL fractions at 800 x g for 5 min at 4 &#176;C. Wash the pooled IEL fractions with 10 ml of HBSS/5% FBS and spin them down for 5 min at 800 x g and 4 &#176;C.</li>
	<li>Resuspend the cells in 8 mL of 44% colloidal silica medium15 and overlay them with 5 ml of 67% colloidal silica medium. Centrifuge the 44%/67.5% cell mixture gradient for 20 min at room temperature and 1,500 x g.</li>
	<li>Collect the IEL cells from the band at the interface. First, remove the top layer of the supernatant (about 5 ml) using 1 ml pipette. Then, harvest IELs at the interphase of the colloidal silica medium gradient.</li>
	<li>Wash the IELs by adding 10 ml of RPMI medium. Spin the cells down for 5 min at 800 x g and 4 &#176;C. Resuspend the IELs in 5 ml of RPMI for step 3.15.</li>
	<li>For the isolation of the lamina propria lymphocytes (LPLs), mince the intestine tissue fragments from step 3.4 into small pieces using scissors (1 mm) and digest the pieces with 10 ml of digestion buffer (0.05 g of collagenase, 0.05 g of DNaseI, and 0.3 g of dispase II in RPMI/5% FBS) for 15-20 min at 37 &#176;C.</li>
	<li>Filter the mixture through a 70 &#181;m cell strainer. The flow-through supernatant contains the released LPL.</li>
	<li>Fully digest the intestine fragments by repeating steps 3.9-3.10 (normally, they must be repeated 3 times). Each time, pool the supernatants containing the LPLs.</li>
	<li>Collect the cells by centrifugation for 5 min at 1,500 x g and room temperature and resuspend the pellets in RPMI/5% FBS. Pass them through a 40 &#181;m cell strainer.</li>
	<li>Resuspend the cell pellets in 10 ml of the 40% fraction of a 40:80 colloidal silica medium gradient and overlay them on 5 ml of the 80% fraction in a 15 ml tube.</li>
	<li>Perform the density gradient separation by centrifugation for 20 min at 1,500 x g and room temperature15.</li>
	<li>Collect the LPLs, as described in step 3.7. Wash them once with 10 ml of PBS and resuspend the pellets in 1 ml of PBS/1% FBS buffer or RPMI medium.</li>
	<li>Count the viable cells (both IELs and LPLs) using 0.4% trypan blue dye/PBS solution.</li>
</ol>
4. Expression of IL-22-tdTomato in Colitis
<ol>
	<li>Prepare single spleen cell suspensions from IL-22-TdTomato mice at a concentration of 1 x 107 cells/ml in sterile PBS, as described in steps 2.1-2.4.</li>
	<li>Purify the CD4+ T cells using the Mouse CD4 Cell Negative Isolation method10. Label them with anti-CD4-APC (clone RM4-5, 0.5 &#181;l/1 x 106 cells in PBS/1% FBS buffer) and anti-CD45RB-FITC (clone 16A, 0.5 &#181;l/1 x 106 cells in PBS/1% FBS buffer) by incubation at 4 &#176;C for 30 min.</li>
	<li>Spin the cells down for 5 min at 500 x g and 4 &#176;C. Wash the cells with 2 ml of PBS/1% FBS buffer and resuspend them in 1 ml of PBS/1% FBS staining buffer.</li>
	<li>Run the labeled T cells on a flow cytometry machine16. Gate the cells on a T-cell population and sort CD4+CD45RBhigh double-positive cells (the wavelength detected at 488 nm and 633 nm lasers).</li>
	<li>Harvest the sorted cells by centrifugation for 5 min at 500 x g and 4 &#176;C. Resuspend the cell pellets in sterile PBS buffer at 1 x 106 cells/ml. Inject the cells (0.5 x 106) into the peritoneum ofeach Rag1-/- recipient mouse.</li>
	<li>Sacrifice the recipient mice under CO2 at various time points. Harvest the mesenteric lymph nodes and the small and large intestines, as described in steps 2.1 and 2.2. Put each mesenteric lymph node and cut-open intestine (paper/intestine/paper sandwich) in 4% paraformaldehyde (PFA; handle under a fume hood) overnight to fix the tissues. Replace the PFA with 18% sucrose for 16-24 hr and then freeze the tissues for sectioning.</li>
	<li>Use the cryostat machine to cut the tissue17,18,19. Warm the tissue sections to room temperature for approximately 60 min and place them in acetone for 10 min at room temperature. Dry them at room temperature for approximately 5 min.</li>
	<li>Add FBS in excess, remove it by pipette, and add anti-RFP at a 1:200 dilution in 0.1% BSA/1x PBS. Incubate the antibody at 37 &#176;C for 60 min.</li>
	<li>Rinse the sections in 1x PBS for 10 min and apply the corresponding secondary antibody (goat anti-rabbit 568) to the tissue, diluted 1:300 in 1% BSA/1x PBS, for 30 min at room temperature.</li>
	<li>Rinse slides in 1x PBS for 10 min, wipe them dry, and add coverslips.</li>
	<li>Visualize all samples with a fluorescent microscope under consistent illumination (RFP: excitation filter 540-580 nm) using the 40X objective.</li>
</ol>

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The authors have nothing to disclose.

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.

<table><tbody><tr><td>RP23-401E11 BAC</td><td>Thermo Fisher Scientific</td><td>RPCI23.C</td><td>Need gene ID: 50929</td></tr><tr><td>NucleoBond BAC 100</td><td>Takara Clontech</td><td>740579</td><td></td></tr><tr><td>PCR SuperMix High Fidelity</td><td>Thermo Fisher Scientific</td><td>10790020</td><td></td></tr><tr><td>PI-SceI</td><td>New England Biolabs</td><td>R0696S</td><td></td></tr><tr><td>SpeI</td><td>New England Biolabs</td><td>R0133S</td><td></td></tr><tr><td>LB Broth</td><td>Thermo Fisher Scientific</td><td>10855-001</td><td>1 L: 10 g SELECT Peptone 140, 5 g SELECT Yeast Extract, 5 g sodium chloride&amp;nbsp;</td></tr><tr><td>Anti-mouse CD3</td><td>eBioscience</td><td>11-0031</td><td></td></tr><tr><td>Anti-mouse CD4</td><td>eBioscience</td><td>17-0041</td><td></td></tr><tr><td>Anti-mouse CD45</td><td>Thermo Fisher Scientific</td><td>MCD4530</td><td></td></tr><tr><td>Anti-mouse CD45RB</td><td>eBioscience</td><td>11-0455</td><td></td></tr><tr><td>Anti-mouse RFP</td><td>Abcam</td><td>Ab62341</td><td></td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red</td><td>Thermo Fisher Scientific</td><td>14175145</td><td>KCl, KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, NaHCO&lt;sub&gt;3&lt;/sub&gt;, NaCl, D-Glucose</td></tr><tr><td>Dnase I</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>ACK lysing buffer</td><td>Thermo Fisher Scientific</td><td>A1049201</td><td></td></tr><tr><td>Percoll</td><td>GE healthcare life sciences</td><td>17-0891-01</td><td></td></tr><tr><td>Collagenase D</td><td>Roche</td><td>11088858001</td><td></td></tr><tr><td>Dispase II (neutral protease, grade II)</td><td>Roche</td><td>4942078001</td><td></td></tr><tr><td>IX70 inverted fluorescence microscope</td><td>Olympus</td><td></td><td>Ask for quote</td></tr><tr><td>Nikon Eclipse 80i microscope</td><td>Nikon</td><td></td><td>Ask for quote</td></tr><tr><td>Dynal shaker</td><td>Electron Microscopy Science</td><td>61050-10</td><td></td></tr><tr><td>FACSAria</td><td>BD Bioscience</td><td></td><td>Ask for quote</td></tr><tr><td>LSRII SORP/flow cytometry</td><td>Becton, Dickinson and Company&amp;nbsp;</td><td></td><td>Ask for quote</td></tr></tbody></table>

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 ...

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

Research

JoVE Journal

Immunology and Infection

8.7K Views.  National Cancer Institute, National Institutes of Health. 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. 

Video: Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37&deg;C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent   a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37&deg;C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.

This procedure describes the detection and isolation of mouse TH17 leukocytes that actively secrete IL-17 upon stimulation.

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

Biology

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.

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 ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

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 ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of cytokines is their pleiotropism, as they are produced by and can affect a multitude of cell types. As such, it is important to understand not only which cells are producing cytokines, but also in which environment they do so, in order to define more specific therapeutics. Here, we describe a method to visualize cytokine production in situ following bacterial infection. This technique relies on imaging cytokine-producing cells in their native environment by confocal microscopy. To do so, tissue sections are stained for markers of multiple cell types together with a cytokine stain. Key to this method, cytokine secretion is blocked directly in vivo before harvesting the tissue of interest, allowing for detection of the cytokine that accumulated inside the producing cells. The advantages of this method are multiple. First, the microenvironment in which cytokines are produced is preserved, which could ultimately inform on the signals required for cytokine production and the cells affected by those cytokines. In addition, this method gives an indication of the location of the cytokine production in vivo, as it does not rely on artificial in vitro re-stimulation of the producing cells. However, it is not possible to simultaneously analyze cytokine downstream signaling in cells that receive the cytokine. Similarly, the cytokine signals observed correspond&#160;only to the time-window during which cytokine secretion was blocked. While we describe the visualization of the cytokine Interferon (IFN) gamma in the spleen following mouse infection by the intracellular bacteria Listeria monocytogenes, this method could potentially be adapted to the visualization of any cytokine in most organs.

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse tissues.

Cytokines are small proteins secreted by cells, mediating cell-cell communications that are crucial for effective immune responses. One characteristic of ...

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 ...

Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various pathological processes, including anti-helminth immunity, tissue repair, thermogenesis, and autoimmune diseases such as asthma and allergic rhinitis (AR). ILC2s permanently reside in peripheral tissues such as the skin, gut, lungs, and nasal cavity; however, there is limited information about their exact functions in nasal mucosal immunity. CD226 is an activating costimulatory molecule, mainly expressed on natural killer (NK) cells, T cells, and inflammatory monocytes. However, whether ILC2s express CD226 or play a role in the pathogenesis of ILC2s-related diseases remains unknown. Here, we established a method to isolate and identify ILC2s from the nasal mucosa and detected CD226 expression on ILC2s obtained from healthy and AR mice. Herein, we describe this protocol for the isolation and identification of ILC2s from mouse nasal mucosa, which will help explore the internal pathological mechanism of immunological disorders in nasal mucosal diseases.

Group 2 innate lymphoid cells (ILC2s), implicated in type 2 inflammation, mainly participate in response to helminth infection, allergic diseases, metabolic homeostasis, and tissue repair. In this study, a procedure to isolate ILC2s from murine nasal mucosa and detect the expression of CD226 is demonstrated.

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various ...

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response against helminth infections, where it plays an important role in the expulsion of the parasite. In a murine model of Nippostrongylus brasiliensis infection, IL-9 is produced mainly by CD4+ T lymphocytes and innate lymphoid cells found in the lung, small intestine, and draining lymph nodes. Given the technical difficulties involved in the intracellular staining of IL-9, as well as the complexity of isolating hematopoietic cells from the small intestine upon infection, there is a pressing need for a comprehensive but straightforward protocol to analyze the expression of IL-9 in different lymphoid and non-lymphoid tissues in this model. The protocol described here outlines the kinetics of IL-9 produced by CD4+ T cells and innate lymphoid cells in the lung and small intestine, the main organs targeted by N. brasiliensis, as well as in the mediastinal and mesenteric lymph nodes, throughout the infection. In addition, it details the number of larvae needed for infection, depending on the cell type and organ of interest. This protocol aims to assist in the standardization of assays to save time and resources by offering the opportunity to focus on the specific cells, organs, and disease stages of interest in the N. brasiliensis infection model.

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9-expressing T and ILC2 cells are induced during N. brasiliensis infection, yet their characterization has been largely overlooked in the infected intestine due to their low frequency and differential kinetics. This protocol describes the isolation of these cells from different target organs and confirmation of their identity via flow cytometry at different infection stages.

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Summary

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Chapters in this video

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

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

Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Isolation and Activation of Murine Lymphocytes

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model