Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Summary

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

Abstract

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

Introduction

Mycobacterial infection has been demonstrated to cause host immune cell death¹. For example, an attenuated strain will trigger apoptosis in macrophages and contain the infection. However, a virulent strain will trigger lytic cell death, causing bacterial dissemination^1,2. Considering the impact these different types of cell death have on host anti-mycobacterial response, a detailed observation of macrophage cell death during mycobacterial infection in vivo is needed.

The conventional methods for measuring cell death are to use dead cell stains, such as Annnex....

Protocol

Zebrafish were raised under standard conditions in compliance with laboratory animal guidelines for ethical review of animal welfare (GB/T 35823-2018). All zebrafish experiments in this study were approved (2019-A016-01) and conducted at Shanghai Public Health Clinical Center, Fudan University.

1. M. marinum Single Cell Inoculum Preparation (Figure 1)

1. Thaw Cerulean-fluorescent M. marinum glycerol stock from -80 °C and inoculate a 7H10 agar plate with 10% (v/v) OADC, 0.25% glycerol and 50 μg/mL hygromycin. Incubate the plate at 32 °C for around 10 days.
2. Sele....

Results

Mycobacterium infection can trigger different host responses based on the routes of infection. In this protocol, zebrafish embryos are infected by intramuscular microinjection of fluorescently labeled bacteria into the midbrain or trunk (Figure 3) and observed by confocal live imaging. Infection via these two routes will locally restrict the infection causing innate immune cell recruitment and subsequent cell death.

Visualizing the details of innate immune cell de.......

Discussion

This protocol describes the visualization of macrophage death during mycobacterial infection. Based on factors such as the integrity of the cell membrane, infection driven cell death can be divided into apoptosis and lytic cell death^24,25. Lytic cell death is more stressful for the organism than apoptosis, because it triggers a strong inflammatory response ^24,25. Observation of lytic cell death in vivo is.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05% Tween-80	Sigma	P1379
10 mL syringe	Solarbio	YA0552
10% OADC	BD	211886
3-aminobenzoic acid	Sigma	E10521
5 μm filter	Mille X	SLSV025LS
50 μl/ml hygromycin	Sangon Biotech	A600230
7H10	BD	262710
7H9	BD	262310
A glass bottom 35 mm dish	In Vitro Scientific	D35-10-0-N
Agarose	Sangon Biotech	A60015
Confocal microscope	Leica	TCS SP5 II
Enviromental Chamber	Pecon	temp control 37-2 digital
Eppendorf microloader	Eppendorf	No.5242956003
Glass microscope slide	Bioland Scientific LLC	7105P
Glycerol	Sangon Biotech	A100854
Incubator	Keelrein	PH-140(A)
M.marinum			ATCC BAA-535
Microinjection needle	World Precision Instruments	IB100F-4
Microinjector	Eppendorf	Femtojet
Micromanipulator	NARISHIGE	MN-151
msp12:cerulean			Ref.: PMID 25470057; 27760340
Phenol red	Sigma	P3532
PTU	Sigma	P7629
Single concavity glass microscope slide	Sail Brand	7103
Sonicator	SCICNTZ	JY92-IIDN
Spectrophotometer (OD600)	Eppendorf AG	22331 Hamburg
Stereo Microscope	OLYMPUS	SZX10
Tg(mfap4:eGFP)			Ref.: PMID 30742890
Tg(coro1a:eGFP;lyzDsRed2)			Ref.: PMID 31278008
Tg(mpeg1:LRLG;lyz:eGFP)			Ref.: PMID 27424497; 17477879