Migrazione phagosome e velocità misurata in Live primarie macrofagi umani infettati con 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 their migration on microtubules 3,4 towards the cell center to reach a perinuclear location where lysosomes are accumulated.
Many pathogens have been reported to hijack phagosome maturation, including bacteria with intracellular lifestyles that modify the vacuolar environment where they reside 5. The Human Immunodeficiency Virus (HIV)-1 targets macrophages in addition to T cells. As macrophages are resistant to the cytopathic effects of the virus, unlike T cells, they can be considered as a reservoir for the virus. In addition, macrophages infected with HIV-1 show defective phagocytic functions and contribute to the emergence of opportunistic diseases. In particular, severe invasive non-typhoidal Salmonella disease caused by Salmonella Typhimurium ST313 has been prevalent for the last three decades in sub-Saharan African children or adults infected with HIV 6. It has been estimated that the risk of developing tuberculosis is more than 20 times greater in people living with HIV than among those without HIV infection.
For all these reasons, it is important to better define the molecular mechanisms underlying the phagocytic defects in HIV-infected macrophages. We have shown that the uptake of particulate material, opsonized particles, bacteria or fungi, was inhibited in HIV-infected macrophages 7. Given that this inhibition is partial, we then set out to analyze the fate of the internalized phagosomes in HIV infected human macrophages 8. Because phagosome maturation is tightly connected with migration to the cell center and fusion with lysosomes, a defect in phagosomal maturation can be due to modifications of the trafficking modalities in the infected cell. The method described here uses IgG-opsonized Sheep Red Blood Cells (IgG-SRBCs) as a model to target receptor-mediated phagocytosis and in particular receptors for the Fc portion of immunoglobulins (FcR). These particles are easier to image in bright field (BF) than latex beads because extracellular and intracellular SRBCs show different refraction properties 9. To measure the velocity of phagosomes moving towards the nucleus in HIV-infected macrophages, we used a fluorescent virus 10 and set up a simple manual tracking method that is described here. The method does not require advanced programming and simply uses ImageJ. It is amenable to adherent cells and any type of particle or pathogen that can be visualized with bright field microscopy or with fluorescent imaging.
Protocol
Representative Results
Discussion
Questa tecnica ha diversi passaggi critici. In primo luogo, la preparazione di hMDMs e l'infezione da HIV-1 è critico poiché la percentuale di infezione è donatore dipendente. Da notare, abbiamo deciso di utilizzare macrofagi che non sono polarizzati in vitro prima dell'infezione, perché lo stato macrofagi potenzialmente incontrate dal virus in vivo non è stata ben caratterizzata finora. Abbiamo controllato l'espressione di diversi marcatori di superficie, il che indica che i macrofagi…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Dr Jamil Jubrail for reading the manuscript. This work was supported by grants from CNRS, Inserm, Université Paris Descartes, Agence Nationale de la Recherche (2011 BSV3 025 02), Fondation pour la Recherche Médicale (FRM DEQ20130326518 including a doctoral fellowship for GLB) and Agence Nationale de Recherche sur le SIDA et les hépatites virales (ANRS, including a post-doctoral fellowship for CD) to FN. A. Dumas was supported by doctoral fellowships from Université Paris Descartes and Sidaction.
Materials
| Falcon 100mm 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 | Room temperature |
| 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 (10×500 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 50mW 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 x 6.45 µm ; Definition 1392 x 1040 ; 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 |
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