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

Summary

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.

Abstract

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.

Introduction

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 µm 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 ....

Protocol

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

1. 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 or biological samples were carried out under a biosafety cabinet according to biosafety level (BSL) 2 conditions.
   1. Clean the biosafety cabin....

Results

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........

Discussion

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 developed^9,10,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gigasept instru AF	Schülke & Mayr GmbH		4% solution
CD11c-YFP mice	Jackson Laboratories	008829	mice were bred in-house
CK6-ECFP mice	Jackson Laboratories	004218	mice were bred in-house
1x Dulbecco's Phosphate Buffered Saline modified without Calcium Choride and Magnesium Chloride	Sigma	D8537-500ML
10x Dulbecco's Phosphate Buffered Saline modified without Calcium Choride and Magnesium Chloride	Sigma	D1408-500ML
Percoll PLUS	Sigma	E0414-1L	Store at 4°C
Ketamin Labatec	Labatec Pharma	7680632310024	Store at RT, store at 4 °C when in solution of ket/xyl mixture
Rompun 2% (Xylazin)	Bayer	6293841.00.00	Store at RT, store at 4 °C when in solution of ket/xyl mixture
26 G 1 mL Sub-Q BD Plastipak	BD Plastipak	305501
30 G, 0.3 mL BD Micro-Fine Insulin Syringes	BD	324826
Falcon 40 µm Cell Strainer	Corning	352340
2 mL Syringes	BD Plastipak	300185
Microlance 3x 18 G needles	BD	304622
Introcan Safety 20 G (catheter)	Braun	4251652.01
6 Well Cell Culture Cluster	Costar	3516
RPMI medium 1640 + HEPES (1x)	ThermoFisher Scientific	42401-018	Store at 4°C
Liberase TL Research Grade	Roche	5401020001	Store at -20 °C / collagenase (I and II) mixture
DNAse I	Amresco (VWR)	0649-50KU	Store at -20 °C
CellTrace Violet stain	ThermoFisher Scientific	C34557	Store at -20 °C
EDTA	Sigma	EDS-500G
Fetal Bovine Serum	Gibco	10270-106	Store at -20 °C
PE-10 Micro Medical Tubing	2Biological Instruments SNC	#BB31695-PE/1
Surgical Plastic Tape	M Plast
Viscotears	Bausch & Lomb		Store at RT
Plasticine	Ohropax
High Tolerance Glass Coverslip 15 mm Round	Warner Instruments	64-0733
SomnoSuite Portable Animal Anesthesia System	Kent Scientific	SS-01
Nuvo Lite mark 5	GCE medline	14111211
MiniTag (gaseous anesthesia and heating bench)	Tem Sega
SURGICAL BOARD	University of Bern
TrimScope II Two-photon microscope	LaVision Biotec
Chameleon Vision Ti:Sa lasers	Coherent Inc.
25X NA 1.05 water immersion objective	Olympus	XLPLN25XWMP2
The Cube&The Box incubation chamber and temperature controller	Life imaging Services
Imaris 9.1.0	Bitplane		Imaging software
GraphPad Prism 7	GraphPad		Statistical software