Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Summary

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

Abstract

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.

The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

Introduction

Macrophages play a major role in the innate immune system and in homeostasis. They are professional phagocytes that internalize and eliminate pathogens and debris by a process called phagocytosis ^1,2. The phagosome, the closed compartment that forms after the engulfment of particulate material, undergoes a series of fusion and fission events with endocytic compartments, leading to a degradative compartment called the phagolysosome. This compartment has an acidic pH, due to the acquisition of proton-pumping v-ATPases, contains hydrolytic enzymes and is enriched in lysosomal-associated membrane proteins (LAMPs). The maturation of phagosomes is accompanied by ....

Protocol

The protocol has to be carried out in strict accordance with national and international legislations and local regulations. Blood from healthy donors that gave their consent to donate blood for research purposes has been obtained from Blood Transfusion Centers with which the Institutions have signed agreement. Special protections must be taken when using human blood. Experiments with HIV-1 must be performed in a biosafety level 3 or 2 (BSL-3 or 2) laboratory according to local legislation.

1. Preparation of Human Monocyte-derived Macrophages (hMDMs) by Density Gradient Centrifugation and Selection by Adhesion

1. Start with fresh bl....

Results

FcR-mediated phagocytosis by HIV-1-infected and non-infected hMDMs is described here using IgG-opsonized SRBCs as model targets (Figure 1). The critical steps of this protocol are the preparation of hMDMs and the infection with HIV-1. Indeed, the yield and quality of differentiated macrophages varies among donors, as well as the infection rate with efficiencies in the range of 10-40%. In addition, the preparation of IgG-opsonized-SRBCs is also important to avoid damaging .......

Discussion

This technique has several critical steps. Firstly, the preparation of hMDMs and their infection by HIV-1 is critical because the percentage of infection is donor dependent. Of note, we have decided to use macrophages that are not polarized in vitro prior to infection, because the status of macrophages potentially encountered by the virus in vivo has not been well characterized so far. We checked the expression of several surface markers, indicating that the macrophages are neither M1 nor M2, and verifi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Falcon 100 mm TC-Treated Cell Culture Dish	Corning	353003	For viral production
Glass Bottom Dishes 35 mm uncoated 1.5	MatTek corporation	P35G-1.5-14-C Case	For acquisition
Falcon Tissue Culture Plates 6-well	Thermo Fischer Scientific	Corning. Inc. 353934	For human monocyte-derived macrophages
Ficoll-Plaque PLUS	Dominique Dutscher	17-1440-03	a neutral, highly branched, high-mass, hydrophilic polysaccharide in solution for density centrifugation
DPBS, no calcium, no magnesium	Thermo Fischer Scientific	14190-094	RT
Dulbecco's Modified Eagle Medium (DMEM) 1x, liquid (High Glucose)	GIBCO, Molecular probes	31966-021	Conserved at 4 °C ; for HEK cells culture
RPMI 1640 medium GLUTAMAX  Supplement	Life technologies	61870-010	Conserved at 4 °C; for hMDMs culture
Fœtal Calf Serum (FCS)	Eurobio	CVFSVF0001	Conserved at -20 °C ; decomplemented
Penicillin-Streptomycin (10,000 U/ml)	Thermo Fischer Scientific	15140-122	Conserved at -20 °C ; for hMDMs culture
RPMI 1640 medium, no phenol red (10x 500 ml)	Life technologies	11835-105	Warm in 37 °C water bath before use ; for phagocytosis assay
FuGENE6 Transfection Reagent	Promega	E2692	Conserved at 4 °C ; for viral production
Sheep red blood cells (SRBCs)	Eurobio	DSGMTN00-0Q	Conserved in Alsever buffer at 4 °C before use
Anti-sheep red blood cells IgG	MP Biomedicals	55806	Conserved at 4 °C
Bovine Serum Albumin heat shock fraction, pH 7, ≥98%	Sigma	A7906	Conserved at -20 °C
Inverted microscope DMI600	Leica
Confocal Spinning Disk Unit  CSU-X1M1	Yokogawa
491 nm 50 mW laser	COBOLT CALYPSO
HCX PL APO CS Objectif	Leica		Objective lens ; Magnification 100X ; Numerical aperture 1.40 ; Immersion oil
CoolSnap HQ2 (FireWire) Camera	Photometrics		Pixel size 6.45 µm x 6.45 µm ; Definition 1,392 x 1,040 ; Encoding the image in 14 Bit
Metamorph 7.7.5 software	Molecular Devices		For the control of the microscope
GraphPad Prism software			For the statistics analysis