This work was supported by the Swiss National Foundation (SNF) grants (176124, 145038, and 148183), the European Commission Marie Curie Reintegration Grant (612742), and the SystemsX.ch for a grant to D.U.P. (2013/124).

All animal procedures involving mice were performed in accordance with the Swiss Federal Veterinary Office guidelines and animal protocols were approved by the local veterinarian authorities.
1. Influenza Infection of CD11c-YFP Mice
<ol>
	<li>Biosafety 
	NOTE: The mouse adapted strain of influenza A/Puerto Rico/8/34 H1N1 (PR8) was grown in fertilized eggs, purified and titrated as previously described21. All the steps involving infected animals o...

<ol>
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The authors have nothing to disclose.

This work presents a detailed protocol for the generation of 4D images showing the migration of adoptively transferred neutrophils and their interactions with DC during an influenza infection in the mouse trachea. The described 2P-IVM model will be relevant to study immune cell dynamics during an infection in the airways.
Recently, several models based on the visualization of cell dynamics in the airways have been developed9,10,</...

Two-photon intravital microscopy (2P-IVM) is an effective technique for real time imaging of cell-to-cell interactions as they occur in their natural environment1. One of the main advantages of this method is that it allows the study of cellular processes at a greater specimen depth (500 &#181;m to 1 mm) compared with other traditional imaging techniques2. At the same time, the use of two low-energy photons generated by the two-photon laser minimizes the tissue photo-damage typically associated with the image acquisition process2. During the last decade, 2P-IVM has been applied to study different types of cell-cell interactions in several disciplines3,4,5. These studies have been especially relevant to investigate immune cells, which are characterized by their high dynamism and the formation of prominent contacts following the signals generated by other cells and the environment. 2P-IVM has been also applied to study the interactions between pathogen and host6. Indeed, it has been previously shown that some pathogens can alter the type and duration of the contacts between immune cells, hampering, as a result, the immune response7.
The airway mucosa is the first site in which the immune response against airborne pathogens is generated8. Therefore, in vivo analysis of pathogen-host interactions in this tissue is critical to understand the initiation of the host defense mechanisms during infection. However, 2P-IVM of the airways is challenging mainly due to the artifacts produced by the breathing cycle of the animal, which compromises the process of image acquisition. Recently, different surgical models have been described for imaging murine trachea9,10,11,12 and lungs13,14,15,16. Tracheal 2P-IVM models represent an excellent set-up to visualize the initial phase of the immune reaction in the upper airways, while lung-alveoli 2P-IVM models are more suitable to study the late phase of infections. The developed lung models present a limitation associated with the presence of air-filled alveoli, which restrict the optical penetration of the laser and make the mucosal layer of the intrapulmonary airways inaccessible for in vivo imaging17. Conversely, the structure of the trachea, formed by a continuous epithelium, facilitates image acquisition.
Here, we present a protocol that includes a detailed description of the steps required to perform influenza infection, surgical preparation of the animals, and 2P-IVM of the trachea. In addition, we describe a specific experimental set-up for the visualization of neutrophils and dendritic cells (DC), two immune cell types that play an important role as mediators of the defense mechanism against influenza virus18,19. Finally, we describe a procedure to analyze neutrophil-DC interactions. These contacts have been shown to modulate DC activation and, subsequently, to affect the immune responses against pathogens20.

<table>
	<tbody>
		<tr>
			<td>Gigasept instru AF</td>
			<td>Sch&#252;lke &#38; Mayr GmbH</td>
			<td></td>
			<td>4% solution</td>
		</tr>
		<tr>
			<td>CD11c-YFP mice</td>
			<td>Jackson Laboratories</td>
			<td>008829</td>
			<td>mice were bred in-house</td>
		</tr>
		<tr>
			<td>CK6-ECFP mice</td>
			<td>Jackson Laboratories</td>
			<td>004218</td>
			<td>mice were bred in-house</td>
		</tr>
		<tr>
			<td>1 X Dulbecco&#39;s Phosphate Buffered Saline modified without Calcium Choride and Magnesium Chloride</td>
			<td>Sigma</td>
			<td>D8537-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>10 X Dulbecco&#39;s Phosphate Buffered Saline modified without Calcium Choride and Magnesium Chloride</td>
			<td>Sigma</td>
			<td>D1408-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll PLUS</td>
			<td>Sigma</td>
			<td>E0414-1L</td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Ketamin Labatec</td>
			<td>Labatec Pharma</td>
			<td>7680632310024</td>
			<td>Store at RT, store at 4&#176;C when in solution of ket/xyl mixture</td>
		</tr>
		<tr>
			<td>Rompun 2% (Xylazin)</td>
			<td>Bayer</td>
			<td>6293841.00.00</td>
			<td>Store at RT, store at 4&#176;C when in solution of ket/xyl mixture</td>
		</tr>
		<tr>
			<td>26 G 1 mL Sub-Q BD Plastipak</td>
			<td>BD Plastipak</td>
			<td>305501</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G 0,3 mL BD Micro-Fine Insulin Syringes</td>
			<td>BD</td>
			<td>324826</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 40 &#181;m Cell Strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Syringes</td>
			<td>BD Plastipak</td>
			<td>300185</td>
			<td></td>
		</tr>
		<tr>
			<td>Microlance 3 18 G needles</td>
			<td>BD</td>
			<td>304622</td>
			<td></td>
		</tr>
		<tr>
			<td>Introcan Safety 20G (catheter)</td>
			<td>Braun</td>
			<td>4251652.01</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Cell Culture Cluster</td>
			<td>Costar</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640 + HEPES (1X)</td>
			<td>ThermoFisher Scientific</td>
			<td>42401-018</td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Liberase TL Research Grade</td>
			<td>Roche</td>
			<td>5401020001</td>
			<td>Store at -20&#176;C / collagenase (I and II) mixture</td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Amresco (VWR)</td>
			<td>0649-50KU</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>CellTrace Violet stain</td>
			<td>ThermoFisher Scientific</td>
			<td>C34557</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>EDS-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>PE-10 Micro Medical Tubing</td>
			<td>2Biological Instruments SNC</td>
			<td>#BB31695-PE/1</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Plastic Tape</td>
			<td>M Plast</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Viscotears</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>Store at RT</td>
		</tr>
		<tr>
			<td>Plasticine</td>
			<td>Ohropax</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High Tolerance Glass Coverslip 15mm Round</td>
			<td>Warner Instruments</td>
			<td>64-0733</td>
			<td></td>
		</tr>
		<tr>
			<td>SomnoSuite Portable Animal Anesthesia System</td>
			<td>Kent Scientific</td>
			<td>SS-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuvo Lite mark 5</td>
			<td>GCE medline</td>
			<td>14111211</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniTag (gaseous anesthesia and heating bench)</td>
			<td>Tem Sega</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SURGICAL BOARD</td>
			<td>University of Bern</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrimScope II Two-photon microscope</td>
			<td>LaVision Biotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chameleon Vision Ti:Sa lasers</td>
			<td>Coherent Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25X NA 1.05 water immersion objective</td>
			<td>Olympus</td>
			<td>XLPLN25XWMP2</td>
			<td></td>
		</tr>
		<tr>
			<td>The Cube&#38;The Box incubation chamber and temperature controller</td>
			<td>Life imaging Services</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris 9.1.0</td>
			<td>Bitplane</td>
			<td></td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>GraphPad Prism 7</td>
			<td>GraphPad</td>
			<td></td>
			<td>Statistical software</td>
		</tr>
	</tbody>
</table>

imaging cell interaction in tracheal mucosa during influenza virus infection using two-photon intravital microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this work, we described a detailed protocol to study in vivo the motility and the interactions between neutrophils and DC during influenza infection in murine trachea (Figure 3A). To this purpose, we isolated CFP+ neutrophils (92% purity; Figure 3B) from CK6-ECFP mice and we adoptively transferred them into a CD11c-YFP mouse infected with influenza. After that, we performed 2P-IVM of the trachea at day 3 p....

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Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

This method can help answer key questions in the study of immune responses to airborne pathogens about the dynamics of immune cells'interactions during respiratory infections. The main advantage of this technique is the ability to obtain bright and stable 4D images of the tracheal epithelium throughout the simple surgery. Though this method can provide insight into the neutrophil and the lytic cell interactions during influenza infection, it can also be applied to the study of different immune cells or airborne pathogens.Generally, individuals new to this method will struggle with maintaining a stable and minimal inflamed surgery. Moreover, image analysis can be tricky due to the presence of heart defects. For intranasal influenza infection, place an anesthetized six-to eight-week-old CD11c-YFP mouse in the supine position, and confirm deep sedation by lack of response to toe pinch.Placing the pipette close to the left nostril, dispense small droplets into the nasal cavity approximately 20 seconds apart, until the mouse has inhaled the entire 15 microliter volume of PR8 inoculate. After two to five minutes, deliver 15 microliters of virus into the right nostril in the same manner. When all of the inoculum has been delivered, confirm that the mouse is breathing normally, and place the animal in its cage in lateral decubitus with monitoring until full recovery.Three days post-infection, remove both femora and tibiae from a transgenic CFP-expressing mouse, and gently clean the bones with forceps. Cut the bone epiphysis and use a one-milliliter syringe equipped with a 26 gauge needle to flush the bone marrow from each bone with sterile cold PBS into a 50-milliliter tube on ice. When all of the bone marrow has been harvested, aspirate the marrow through the needle tip several times and filter the dissociated cell suspension through a 40-micrometer strainer.Collect the cells by centrifugation, and resuspend the pellet in two milliliters of cold PBS. Next, carefully layer two milliliters of each of three Percoll gradients in a 15-milliliter tube from most to least concentrated. Then carefully add two milliliters of bone marrow cell suspension onto the top gradient layer for centrifugal density gradient separation.At the end of the centrifugation, carefully remove the band from the 64 and 72%gradient interface and wash the neutrophils in cold PBS. Then resuspend the pellet in 100 microliters of fresh PBS on ice for counting, and resuspend the cells at a five times 10 to the sixth cells per 100 microliters of PBS concentration for intravenous injection into the inoculated CD11c-YFP recipient animals. 12 hours after adoptive transfer, place the anesthetized recipient animal on a customized surgical board over a 37-degree Celsius heated surface, and remove the hair from the neck of the mouse by shaving and a depilatory cream.Place the mouse above the plastic mouse positional with the head outside of the positional at an angle that facilitates intubation of the trachea, and secure the forelimbs, paws, and the tail with surgical tape. Apply ointment to the eyes and disinfect the exposed skin. Next, make a one-centimeter incision along the medial axis of the neck between the upper chest and the line passing through the lower point of the mandible, and laterally move the skin patches and the salivary glands to visualize the trachea.Use forceps to carefully dissect the trachea, intubate the mouse with a catheter, and immediately start the artificial ventilation. Use a surgical hook connected to a rod on the surgical board to fix the catheter height and orientation, and expose the trachea up to the chin. Encircle the trachea with petroleum jelly and hydrate the trachea with a few drops of warm PBS.Then glue a coverslip onto a metal holder, and screw the holder to the XYZ translator to allow the coverslip to be placed on top of the surgical preparation. It is very critical to properly expose and extend the trachea, and to lightly press it with the coverslip, to maintain the tissue stable long enough for the imaging. For time-lapse imaging in vivo, place the surgical board inside the 37-degree Celsius incubation chamber of the two-photon microscope, and add a drop of water to the coverslip.After centering the setup and focusing on the tracheal tissue, set the scanning frequency to 800 hertz with 520 by 520 pixels, a field of view of 440 by 440 micrometers, and a line average of one. Set the titanium sapphire lasers to the appropriate wavelength and power percentage according to the excitation fluorophores, and set up the 3D and time-lapse acquisition mode with simultaneous excitation. Then define a range of 50 micrometers along the z-axis with a step size of three micrometers, and record images every 30 seconds for 30 minutes.Following this experimental setup, stable 4D images can be captured in situ within the infected trachea over a 30-minute tracking period. Analysis of the 4D images reveals significant differences between dendritic cell and recruited neutrophil movement with a significantly faster speed demonstrated by the latter. The complex morphology of the dendritic cells causes frequent errors in cell tracking, which in turn produces tracks with a decreased duration and an increased variance in the measured directional behavior.Therefore, a robust metric was created for measuring the directionality by considering the track duration, revealing a significant difference in the directionality of neutrophils compared to dendritic cells. In addition, computation of the distance between the neutrophils and the dendritic cells allows the detection and analysis of their contacts over time, with some neutrophils in this representative experiment forming multiple brief contacts with dendritic cells and some not forming any contact with dendritic cells during the imaging period. Moreover, study of the average trend of the distance between neutrophils and dendritic cells over time facilitates the study of the overall positioning of the cells, while investigation of the trend in specific cells allows characterization of the behavior of each individual cell.Knowing this procedure, other methods like flow cytometry of the trachea can be performed to assess the quantification and characterization of different immune cells'recruitments in infected trachea. Don't forget that working with any influenza virus strain, even if mouse-adapted, can be hazardous, and that therefore, every procedure should be performed under biosafety level two conditions.

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Immunology and Infection

8.2K visualizações.  Università della Svizzera italiana (USI). This method can help answer key questions in the study of immune responses to airborne pathogens about the dynamics of immune cells'interactions during respiratory infections. The main advantage of this technique is the ability to obtain bright and stable 4D images of the tracheal epithelium throughout the simple surgery. Though this method can provide insight into the neutrophil and the lytic cell interactions during influenza infection, it can also be applied to the study of different immun...