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Immunology and Infection

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Published: July 29th, 2021

DOI:

10.3791/62459

1Laboratory of Host-Parasite Interaction and Epidemiology, Gonçalo Moniz Institute, 2National Institute of Science and Technology of Tropical Diseases - National Council for Scientific Research and Development (CNPq)
* These authors contributed equally

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).

Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the genus Leishmania, resulting in a broad spectrum of clinical manifestations in the vertebrate host, including cutaneous leishmaniasis, mucocutaneous leishmaniasis and visceral leishmaniasis1. The World Health Organization (WHO) estimates that over one billion people are at risk, with more than one million new cases reported per year2.

Leishmania spp. are obligate intracellular protozoans that survive inside host cells, including monocytes, macrophages and dendritic cells3<....

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Cells were obtained from healthy donors following the approval of procedures by the National Research Ethics Committees (ID: 94648218.8.0000.0040).

1. Cell cultures

  1. Human monocyte-derived macrophages
    NOTE: To obtain human monocyte-derived macrophages for in vitro differentiation into macrophages, collect blood from healthy donors and purify peripheral blood mononuclear cells (PBMC) as described by D. English and B. R. Andersen21.
    1. .......

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This report aims to evaluate the early events occurring during the phagocytosis of L. braziliensis isolated from patients presenting L. braziliensis-LCL or L. braziliensis-DL form of CL. Using confocal microscopy, we investigated the main events associated with parasites' phagocytosis: binding, internalization, and phagosome maturation. We first evaluated the L. braziliensis-LCL or L. braziliensis-DL binding and phagocytosis by human monocyte-derived macrophages. The data .......

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Leishmania-macrophage interaction is a complex process and involves several steps that can influence disease development5. To better understand the mechanisms involved in the interaction of unopsonized Leishmania and host cells, we have described a protocol that employs confocal fluorescence microscopy to assess phagocytosis from early to late stages of Leishmania infection. The use of fluorescence techniques involving two or more fluorophores to investigate cell biology.......

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We thank Gonçalo Moniz Institute, Fiocruz Bahia, Brazil and the department of microscopy for assistance. This work was supported by INOVA-FIOCRUZ number 79700287000, P.S.T.V. holds a grant for productivity in research from CNPq (305235/2019-2). Plasmids were kindly provided by Mauricio Terebiznik, University of Toronto, CA. The authors would like to thank Andris K. Walter for English language revision and manuscript copyediting assistance.

....

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Name Company Catalog Number Comments
2-mercaptoethanol Thermo Fisher Scientific 21985023
AlexaFluor 488-conjugated goat anti-rabbit IgG Thermo Fisher Scientific Tem varios no site
anti-LC3 antibody Novus Biologicals NB600-1384
Bovine serum albumin (BSA) Thermo Fisher Scientific X
CellStripper Corning 25-056-CI
CellTracker Red (CMTPX) Dye Thermo Fisher Scientific C34552
Centrífuga Thermo Fisher Scientific
Ciprofloxacin Isofarma X
CO2 incubator Thermo Fisher Scientific X
Confocal fluorescence microscope (Leica SP8) Leica Leica SP8
Fetal Bovine Serum (FBS) Gibco 10270106
Fluorescence microscope (Olympus Lx73) Olympus Olympus Lx73
Gentamicin Gibco 15750045
Glutamine Thermo Fisher Scientific 35050-061
HEPES (N- 2-hydroxyethyl piperazine-N’-2-ethane-sulfonic acid) Gibco X
Histopaque Sigma 10771
M-CSF Peprotech 300-25
NH4Cl Sigma A9434
Normal goat serum Sigma NS02L
Nucleofector 2b Device Lonza AAB-1001
Nucleofector solution Lonza VPA-1007
Paraformaldehyde Sigma 158127
Phalloidin Invitrogen A12379
Phorbol myristate acetate (PMA) Sigma P1585
Phosphate buffer solution (PBS) Thermo Fisher Scientific 10010023
ProLong Gold Antifade kit Life Technologies P36931
Roswell Park Memorial Institute (RPMI) 1640 medium Gibco 11875-093
Saponin Thermo Fisher Scientific X
Schneider's Insect medium Sigma S0146
Sodium bicarbonate Sigma S5761
Sodium pyruvate Sigma S8636
Triton X-100 Sigma X

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