Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Summary

We describe a method to visualize GFP-labeled γδ 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.  

Abstract

Intraepithelial lymphocytes expressing γδ T cell receptor (γδ IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the γδ T cell receptor, our understanding of the regulation of γδ IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating γδ IEL function and the responsiveness of these cells to the local microenvironment. Although γδ 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 γδ 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.

Introduction

Intraepithelial lymphocytes (IEL) are located within the intestinal epithelium, and are found both along the basement membrane and between adjacent epithelial cells in the lateral intercellular space¹. There is approximately one IEL for every 5-10 epithelial cells; these IELs serve as sentinels to provide immune surveillance of the large expanse of the intestinal epithelial barrier². IELs expressing the γδ T cell receptor (TCR) comprise up to 60% of the total IEL population in the murine small intestine. Studies in γδ T-cell-deficient mice demonstrate a largely protective role of these cells in response to intestinal injury, inflammation and infection^3,4,5. Despite the generation of the Tcrd knockout mouse⁶, our understanding of γδ IEL biology remains limited due in part to the fact that ligands recognized by the γδ TCR have yet to be identified⁷. As a result, the lack of tools to study this cell population has made it difficult to investigate the role of γδ TCR activation and function under physiological and pathological conditions. To fill this gap, we have developed live imaging techniques to visualize γδ IEL migratory behavior and interactions with neighboring enterocytes as a means to provide additional insight into γδ IEL function and responsiveness to external stimuli in vivo.

Over the last decade, intravital imaging has significantly expanded our understanding of the molecular events involved in multiple facets of intestinal biology, including epithelial cell shedding⁸, regulation of epithelial barrier function^9,10, myeloid cell sampling of luminal contents^11,12, and host-microbe interactions^{11,13,14,15,16}. In the context of IEL biology, the use of intravital microscopy has shed light on the spatiotemporal dynamics of IEL motility and the factors mediating their surveillance behavior^13,14,15,16. The development of TcrdH2BeGFP (TcrdEGFP) reporter mice, which labels γδ IELs by nuclear GFP expression¹⁷, revealed that γδ IELs are highly motile within the epithelium and exhibit a unique surveillance behavior that is responsive to microbial infection^17,13,14. Recently, another γδ T cell reporter mouse was developed (Tcrd-GDL) which expresses GFP in the cytoplasm to allow visualization of the entire cell¹⁸. Similar methodology has been used to investigate the requirement of specific chemokine receptors, such as G protein-coupled receptor (GPCR)-18 and -55, on the dynamics of IEL motility^19,20. In the absence of a cell-specific reporter, fluorescent conjugated antibodies against CD8α were used to visualize and track IEL motility in vivo^19,20. Although two-photon laser scanning microscopy is commonly used for intravital imaging, the use of spinning disk confocal laser microscopy provides unique advantages to capture high speed and high-resolution multi-channel images with minimal background noise. This technology is ideal to elucidate the spatiotemporal dynamics of immune/epithelial interactions within the complex microenvironment of the intestinal mucosa. Moreover, through the use of various transgenic and/or knockout mouse models, these studies can provide insight into the molecular regulation of intestinal immune and/or epithelial cell function.

Protocol

All studies were conducted in an Association of the Assessment and Accreditation of Laboratory Animal Care (AALAC)-accredited facility according to protocols approved by Rutgers New Jersey Medical School Comparative Medicine Resources.

1. Mouse Preparation

NOTE: The following procedure, including animal preparation and surgery, will take 30–40 min. Prior to the surgery, turn on the microscope and warm up the enclosed incubator on the microscope to 37 °C.

1. Perform experiments on 8–10 weeks old TcrdEGFP mice on a C57BL/6 background, which were obtained from Bernard Malissen (INSERM, Paris, France). According to IACUC-approved procedure, anesthetize the mouse by i.p. injection of ketamine (120 mg/kg) and xylazine (10 mg/kg) based on the weight of the animal. Evaluate the level of anesthesia by respiration rate (55–65 breaths per minute)²¹, toe pinch and palpebral reflex.
2. Once surgical plane anesthesia is reached, secure the hind legs of the mouse using tape to a heating pad to maintain body temperature, which can be monitored by rectal thermometer. If there are signs that the anesthesia is wearing off (e.g., increased respiration rate, blinking and/or eye muscle twitching response to palpebral reflex), re-administer 50 μL of ketamine/xylazine at a time until the mouse is fully sedated.
3. Prepare the nuclear dye by diluting 50 µL of Hoechst 33342 (10 mg/mL) with 315 µL of 0.9% saline; the final concentration is 37.5 mg/kg based on the weight of the mouse (200 µL approximate volume). Once the mouse is anesthetized, slowly inject the Hoechst using a tuberculin syringe through the retro-orbital venous sinus.
   NOTE: Wait 1–2 min before proceeding to the next step to allow the mouse to acclimate to the change in circulating volume following retro-orbital injection.
4. Place a small amount of lubricant over the eyes to prevent them from drying out.

2. Mouse Surgery: Laparotomy to Expose Intestinal Mucosa

1. Make a 2 cm vertical incision through skin and peritoneum along the midline of the lower abdomen to externalize the region of intestine to be imaged.
2. Using angled forceps, carefully pull the cecum out of the peritoneal cavity and identify the area of small intestine to be imaged. Be careful not to tear mesenteric blood vessels during this process. To minimize potential damage or bleeding, avoid grabbing or pinching the intestine with the forceps.
3. Locate a suitable region of small intestine (2–3 cm) for imaging that contains minimal fecal contents. Carefully return the cecum and remaining small intestine into the peritoneal cavity, while leaving the segment of interest externalized.
   NOTE: Avoid puncturing the cecum or disrupting the mesenteric vasculature during this step.
4. Place two pairs of forceps on either side of the underlying mesentery between blood vessels, and gently rub the tips of the forceps together to create a hole in the membrane (Figure 1)²². This will allow the needle to pass through the mesentery when closing the peritoneal cavity beneath the intestine.
5. Close the incision while keeping the loop of intestine externalized. Using angled forceps and a curved, taper point needle attached to a 5 cm suture, penetrate one side of the peritoneum, go through the hole made in the previous step, and up through the other side of the peritoneal lining. Place one suture at the top, and another near the bottom of the incision.
   NOTE: Avoid damaging the vasculature during this step.
6. Repeat this process to close the skin beneath the intestinal loop, by placing one suture in the middle of the incision, between the previous sutures in the underlying peritoneum.
   NOTE: It is important to only externalize the area that is to be imaged; this will reduce extraneous motion during imaging. Be sure to avoid tying off the mesenteric arteries.
7. Use an electrocautery to make a line of perforation along the anti-mesenteric border. Immediately after cauterization, apply a few drops of water to the surface of the intestine to prevent additional heat-induced tissue damage. Blot with a Kimwipe to remove residual water.
8. Cut a small horizontal slit at the distal edge of the cauterized tissue using Vannas scissors and proceed to cut along the length of the cauterized line toward the proximal end of the externalized tissue segment (approximately 1.5 cm in length) to expose the mucosal surface.
   NOTE: If necessary, use forceps to gently remove any excess fecal content remaining on the exposed mucosal surface.
9. Cover the abdomen of the mouse with a moist Kimwipe to prevent the tissue from becoming dehydrated. Transport the mouse to the microscope in a covered vessel.

3. Image Acquisition by Spinning Disk Confocal Microscopy

1. Pipette 150 µL of 1 µM AlexaFluor 633 in HBSS onto the glass coverslipped bottom of a 35 mm dish. Position the mouse so that the opened mucosal surface directly contacts the coverslip.
2. Tilt the head of the microscope back and place the mouse on the coverslipped dish on the imaging stage in a pre-warmed incubator. Alternatively, cover the mouse by a heating blanket to maintain body temperature.
3. Launch imaging software. The excitation intensity and exposure time for each laser should not exceed 10–15 mW or 120–150 ms, respectively, to avoid photobleaching and to minimize the interval between time points. Adjust the frame average to “2” and turn on the EM gain function to reduce background noise. Select 63x objective calibration to ensure the correct measurement of pixel size.
   NOTE: The settings detailed above are meant to provide an initial reference, but will vary depending on individual microscope configurations.
4. Using the 405 nm laser and the 20x air objective, visualize the nuclei to locate a field of villi that lack noticeable movement or drift by eye. Avoid areas of artifactual movement due to respiration, peristalsis or heartbeat.
5. Using the XY scan, record the XY coordinate of the field of interest and switch to the glycerol immersion 63X objective.
6. Confirm that the villi in the selected field are stable by acquiring a live image on the 405 nm channel for up to 1 min.
7. While acquiring a live image on the 405 nm channel, adjust the focus to find the orthogonal plane just beneath the villous tip. Acquire Z-stacks starting from the just below the villous tip epithelium down the villus until it is difficult to resolve the nuclei, about 15–20 µm, using a 1.5 µm step.
   NOTE: When imaging with three channels (405, 488, 640 nm) using the exposure times described above, it is possible to acquire all three channels sequentially at approximately 20 s intervals.
8. Open the software in analysis mode, 3–5 min after beginning the acquisition, to confirm the image stability and γδ IEL motility. Continue to acquire images for 30–60 min for each field of villi. Record 2–3 fields for each mouse. While imaging, monitor the mouse every 5 min.
   NOTE: If the anesthesia begins to wear off, use forceps to gently scruff the neck of the mouse to administer 50 µL of ketamine/xylazine at a time. Continue to monitor anesthesia levels and re-administer when necessary.
9. It is not advisable to image a single mouse for longer than 4 h. Once image acquisition is complete, sacrifice the mice by cervical dislocation followed by bilateral pneumothorax.

4. 4D Analysis of Images

1. Directly import imaging files to 4D rendering software.
2. Create individual Surfaces for IELs and the lumen, then track the surfaces over time using the parameters outlined in Table 1 as an initial reference. For the IELs, enable Split touching objects using the seed point diameter indicated.
3. Use the autoregressive tracking algorithm to determine IEL motility. Although the tracking algorithm is relatively accurate, it is imperative to visually inspect tracks for each individual IEL. If a track is incorrect, correct it using the Disconnect and Connect functions under the Edit Tracks tab.
4. To determine the distance from each IEL to the lumen, select the lumen Surface and perform Distance Transformation function under the Tool menu. When prompted, select Outside Surface. Once calculated, the distance transformation will be displayed as the 4th channel.
5. Filter the Intensity min of the 4th channel to select γδ IELs that are within the lateral intercellular space (LIS). This value should be close to 15 µm, based on the height of a columnar epithelial cell. The percent of total γδ IELs within this range are reported as the frequency of γδ IELs in the LIS.
6. For further analysis, export statistical data including: IEL track speed, track displacement, track straightness and track length. Alternatively, analyze data using the Vantage module. Depending on the experiment, obtain additional metrics from the acquired data, including dwell time within the LIS or IEL/epithelial interactions.

Results

Using intravital imaging of TcrdEGFP reporter mice, we have previously shown that γδ IELs exhibit a dynamic surveillance behavior, in which they patrol the epithelium by migrating along the basement membrane and into the lateral intercellular space (LIS) at steady state (Figure 2, Movie 1).

This approach can also be used to evaluate how the inhibition of specific cell signaling pathways and/or receptors influences γδ IEL migrat...

Discussion

The development of intravital microscopy techniques has provided an unprecedented opportunity to observe the reorganization of subcellular structures^8,9,22, cell-cell interactions^12,25 and cell migratory behavior^13,14,15,16,

Materials

Name	Company	Catalog Number	Comments
35mm dish, No. 1.5 Coverslip	MatTek	P35G-1.5-14-C
Alexa Fluor 633 Hydrazide	Invitrogen	A30634
BD PrecisionGlide Hypodermic needles - 27g	Thermo Fisher Scientific	14-826-48
BD Slip Tip Sterile Syringe - 1 ml	Thermo Fisher Scientific	14-823-434
BD Tuberculin Syringe	Thermo Fisher Scientific	14-829-9
Dissecting scissors	Thermo Fisher Scientific	08-940
Electrocautery	Thermo Fisher Scientific	50822501
Enclosed incubation chamber	OKOLAB		Microscope
Eye Needles, Size #3; 1/2 Circle, Taper Point, 12 mm Chord Length	Roboz	RS-7983-3
Hank's Balanced Salt Solution	Sigma-Aldrich	55037C
Hoechst 33342	Invitrogen	H3570
Imaris (v. 9.2.1) with Start, Track, XT modules	Bitplane		Software
Inverted DMi8	Leica		Microscope
IQ3 (v. 3.6.3)	Andor		Software
Ketamine	Putney		Anesthesia
Kimwipes	VWR	21905-026
McPherson-Vannas scissors 3” (7.5 cm) Long 5X0.15mm Straight Sharp	Roboz	RS-5600
Non-absorbable surgical suture, Silk Spool, Black Braided	Fisher Scientific	NC0798934
Nugent Forceps 4.25” (11 cm) Long Angled Smooth 1.2mm Tip	Roboz	RS-5228
Puralube Vet Ointment	Dechra		Lubricating Eye Ointment
Spinning disk Yokogawa CSU-W1 with a 63x 1.3 N.A. HC PLAN APO glycerol immersion objective, iXon Life 888 EMCCD camera, 405 nm diode laser, 488 nm DPSS laser, 640 nm diode laser	Andor		Confocal system
Xylazine	Akorn		Anesthesia