Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction

Summary

Here we study implications of Leishmania-host interaction by exploring Leishmania-infected dendritic cells migration. The differentiation and infection of dendritic cells, migration analysis, and the evaluation of adhesion complexes and actin dynamics are described. This method can be applied to other host cell migration studies when infected with Leishmania or other intracellular parasite species.

Abstract

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-resolving localized cutaneous lesions to a highly fatal visceral form of the disease. An estimated 12 million people worldwide are currently infected, and another 350 million face risk of infection. It is known that host cells infected by Leishmania parasites, such as macrophages or dendritic cells, can migrate to different host tissues, yet how migration contributes to parasite dissemination and homing remains poorly understood. Therefore, assessing these parasites' ability to modulate host cell response, adhesion, and migration will shed light on mechanisms involved in disease dissemination and visceralization. Cellular migration is a complex process in which cells undergo polarization and protrusion, allowing them to migrate. This process, regulated by actin and tubulin-based microtubule dynamics, involves different factors, including the modulation of cellular adhesion to the substrate. Cellular adhesion and migration processes have been investigated using several models. Here, we describe a method to characterize the migratory aspects of host cells during Leishmania infection. This detailed protocol presents the differentiation and infection of dendritic cells, the analysis of host cell motility and migration, and the formation of adhesion complexes and actin dynamics. This in vitro protocol aims to further elucidate mechanisms involved in Leishmania dissemination within vertebrate host tissues and can also be modified and applied to other cell migration studies.

Introduction

Leishmaniasis, a neglected tropical disease caused by protozoan parasites belonging to the genus Leishmania, results in a wide-ranging spectrum of clinical manifestations, from self-healing localized cutaneous lesions to fatal visceral forms of the disease. It has been estimated that up to one million new leishmaniasis cases arise annually, with a reported 12 million people currently infected worldwide¹. Visceral leishmaniasis (VL), which can be fatal in over 95% of cases when left untreated, causes more than 50,000 deaths annually, affecting millions in South America, East Africa, South Asia, and the Mediterranean region

Protocol

The procedures described herein were approved by the Institutional Review Board of the Gonçalo Moniz Institute (IGM-FIOCRUZ, protocol no. 2.751.345). Blood samples were obtained from healthy volunteer donors. Animal experimental procedures were conducted in accordance with the Ethical Principles in Animal Research adopted by the Brazilian law 11.784/2008 and were approved and licensed by the Ethical Committee for Animal Research of the Gonçalo Moniz Institute (IGM-FIOCRUZ, protocol no. 014/2019).

Discussion

The method described here for evaluating cell migration using the cell culture membrane inserts system allows researchers to study the migratory response of cells in a two-dimensional environment. In this technique, some steps are considered critical. Firstly, the differentiation of human DCs and infection with Leishmania are determinative since the infection rate is donor-dependent. Using more than one donor per experiment and healthy Leishmania cultures will allow for more consistent results. It is al.......

Materials

Name	Company	Catalog Number	Comments
16 Gauge needle	Descarpack	353101
24 well cell culture plate	JET-BIOFIL	J011024
25 Gauge needle	Descarpack	353601
Albumin from bovine serum	Sigma Aldrich	A2153-100G
Ammonium chloride	Sigma Aldrich	A-0171
Anti-mouse IgG, Alexa Fluor 488	Invitrogen	A32723
Anti-mouse IgG, Alexa Fluor 594	Invitrogen	A11032
Anti-rabbit IgG, Alexa Fluor 568	Invitrogen	A11011
CD14 MicroBeads	MACS Myltenyi Biotec	130-050-201
Cell Culture Flask 25cm2	SPL	70125
Cellstripper	Corning	25-056-CI
Confocal microscope	Leica	TCS SP8
Coverslip circles 13mm	Perfecta	10210013CE
Dissecting Forceps	VWR	82027-406
EDTA	Sigma Aldrich	E6758
Falcon tube	KASVI	K19-0051
Fetal Bovine Serum	gibco	16000044
Fluorescence microscope	Olympus	BX51
Glass slide  25,4x76,2mm	Perfecta	200
Hemin bovine	Sigma Aldrich	H2250
Hemocytometer	Perfecta	7302HD
Histopaque® 1077	Sigma Aldrich	10771
MACS buffer	MACS Myltenyi Biotec	130-091-221
Minimum Essential Medium	Gibco	41090093
Mouse anti-Rac1	BD	610650
Paraformaldehyde	Sigma Aldrich	158127
Phalloidin Alexa Fluor 488	Invitrogen	A12379
Phosphate Buffered Saline	ThermoFisher	AM9624
Polycarbonate Membrane Transwell Inserts - Pore size 5.0 µm	Corning	3421
ProLong Gold DAPI kit	Invitrogen	P36931
Rabbit anti-Cdc42	Invitrogen	PA1-092X
Rabbit anti-FAK (pTyr397)	Invitrogen	RC222574
Rabbit anti-paxilin (pTyr118)	Invitrogen	QF221230
Rabbit anti-RhoA	Invitrogen	OSR00266W
Recombinant Human CCL3	R&D Systems	270-LD-010
Recombinant Human GM-CSF	PeproTech	300-03
Recombinant Human IL-4	PeproTech	200-04
Recombinant Human M-CSF	PeproTech	300-25
RPMI 1640 Medium	Gibco	21870076
Saponin	Sigma Aldrich	47036 – 50G – F
Syringe 3 mL	Descarpack	324201
Trypan Blue	Gibco	15250061