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

Published: August 04, 2021

doi:

Yasmin da Silva Luz*¹, Amanda R. Paixão*¹, Carla Polyana O. S. Bernardes, Thaílla Souza da Silva, Natalia Machado Tavares, Claudia I. Brodskyn, Patricia S. T. Veras, Juliana P. B. de Menezes

¹Laboratory of Host – Parasite Interaction and Epidemiology,Gonçalo Moniz Institute

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 region². The main etiological agent of VL in the new world, Leishmania infantum, is transmitted to humans by infected female sandflies during blood-feeding³. These parasites are recognized and internalized by phagocytes, e.g., macrophages and dendritic cells³^,⁴^,⁵. Inside these cells, parasites differentiate into their intracellular forms, known as amastigotes, which will then multiply and be transported via the lymphatic system and bloodstream to different host tissues⁶^,⁷. However, the mechanisms by which Leishmania parasites are disseminated in the vertebrate host, as well as the role played by host cell migration in this process, remain unclear.

Cell migration is a complex process executed by all nucleated cells, including leukocytes⁸. According to the classic cycling model of cell migration, this process involves several integrated molecular events that can be divided into five steps: leading-edge protrusion; adhesion of the leading edge to matrix contacts; contraction of cellular cytoplasm; release of the rear edge of the cell from contact sites; and the recycling of membrane receptors from the rear to the front of the cell⁹.

For cell migration to occur, protrusions must be formed and then stabilized through attachment to the extracellular matrix. Among the different receptor types involved in the promotion of cell migration, integrins are notable. Integrin activation results in migration-related signaling; intracellular signaling then occurs via focal adhesion kinase (FAK) and Src family kinases, in addition to talin, vinculin, and paxillin molecues¹⁰^,¹¹^,¹². The phosphorylation of paxillin by activated kinases, including FAK, leads to the recruitment of effector molecules, which transduces external signals that prompt cell migration. It has been shown that paxillin is an intracellular molecule that is crucial to cell adhesion, actin polymerization, and cell migration processes¹³^,¹⁴^,¹⁵.

The actin cytoskeleton plays a central role in the polarization and migration of phagocytes¹⁶. During cell migration process, protrusions formed due to actin polymerization become stabilized through cell adhesion to the extracellular matrix. This process may be modulated by integrin receptors associated with the actin cytoskeleton¹⁷^,¹⁸^,¹⁹. Several actin-binding proteins regulate the rate and organization of actin polymerization in cellular protrusions²⁰. Studies have shown that RhoA, Rac, and Cdc42 regulate actin reorganization after the stimulation of adherent cells by extracellular factors²¹^,²². During migration, Rac1 and Cdc42 are located at the leading edge of the cell, controlling the extension of lamellipodia and filopodia, respectively, while RhoA, located at the rear of the cell, regulates the contraction of the actomyosin cytoskeleton¹⁵^,²³^,²⁴^,²⁵.

Studies have shown that Leishmania infection modulates host cell functions, such as adhesion to the cellular substrate and migration²⁶^,²⁷^,²⁸^,²⁹^,³⁰^,³¹. Immature DCs reside in peripheral tissues; upon interaction with PAMPS, these cells become activated and migrate to the draining lymph nodes, prompting antigen presentation to T cells. A previous study using a mouse model showed that L. amazonensis infection provokes a reduction in the migration of DCs to draining lymph nodes²⁹. It was also demonstrated that the inhibition of the adhesion process reduced DC migration after infection with L. major³⁰. Nonetheless, the impact of DC migration on parasite dissemination in the host, as well as the mechanisms involved in this process, remain poorly understood.

Here we present a compiled step-by-step protocol to perform an in vitro adhesion and migration assay involving human DCs infected by Leishmania. This method comprises not only the differentiation and infection of DCs, but also permits the analysis of host cell motility and migration, the formation of adhesion complexes, as well as actin dynamics. The presently described in vitro protocol allows researchers to further investigate the mechanisms involved in Leishmania dissemination within vertebrate host tissues and can also be manipulated and applied to other cell migration studies.

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). <p class="…

Representative Results

This protocol described herein enables the evaluation of cell migration and its associated mechanisms, such as actin dynamics and adhesion, thereby providing a tool to determine the migration of Leishmania-infected host cells within the vertebrate host. The results presented here demonstrate that this in vitro assay provides rapid and consistent indications of changes in cellular adhesion, migration, and actin dynamics prior to in vivo experimentation. First, cells w…

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…

Divulgations

The authors have nothing to disclose.

Acknowledgements

This work was supported by Bahia Research Support Foundation (Fapesb), grant number 9092/2015. The authors acknowledge CNPq, Capes and Fapesb for financial support via scholarships. The authors would like to thank Andris K. Walter for critical analysis, English language revision and manuscript copyediting assistance.

Materials

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 25,4×76,2mm	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

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