Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Summary

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.

Abstract

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.

Introduction

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 cells^3<....

Protocol

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. .......

Discussion

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.......

Materials

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