Evaluation of Extracellular Vesicle Function During Malaria Infection

Summary

In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

Abstract

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host. EVs are efficiently taken up by cells in which they modulate their function. Here we discuss strategies to address the role of EVs in parasite-host interactions. First, we describe a straightforward method for labeling and tracking EV internalization by endothelial cells, using a green cell linker dye. Second, we report a simple way to measure permeability across an endothelial cell monolayer by using a fluorescently labeled dextran. Finally, we show how to investigate the role of small non-coding RNA molecules in endothelial cell function.

Introduction

According to the World Health Organization, there were 212 million new cases of malaria worldwide in 2015 and approximately 429,000 people died, mainly children under five years of age¹. The mechanisms leading to severe disease, which is often associated with vascular dysfunction, remain ill-defined². Plasmodium-iRBCs secrete small bi-lipid membrane spheres known as extracellular vesicles (EVs). It is known that these EVs are potentially relevant to the infection process and to the host immune response to infection; however, little is known about the exact function of these small vesicles during malaria infectio....

Protocol

Human RBCs were obtained from the blood of healthy donors, in accordance with the guidelines of Swissethics (swissethics.ch).

NOTE: P. falciparum parasite cultures (3D7) and EV production were previously described in Mbagwu, et al.¹¹ Because P. falciparum is a human pathogen, consult the local regulations for handling. The cultures should be kept sterile the entire time.

1. Fluorescence Labeling of EVs

NOTE: The following procedure takes advantage of the labeling technology to stably incorporate a green fluorescent dye (PKH67) with large....

Results

Here, we describe protocols to investigate the interactions of EVs with host cells. The uptake of fluorescently labeled EVs is monitored by confocal microscopy (Figure 1). Endothelial cells efficiently take up EVs, however the incubation time with EVs can be optimized to track the uptake. For a better localization of EVs inside the cells, stain actin with phalloidin. Next, we use a filter membrane on top of which a monolayer of endothelial cells grows. Rhodam.......

Discussion

Several parasites, including Toxoplasma, Trypanosoma, Leishmania, and Trichomonas trigger the release of EVs by the infected host cell. Depending on the pathogens, the released EVs can modulate the host immune response or mediate cellular communication between the parasites⁶. Yet, there is little evidence suggesting how these small vesicles contribute to malaria disease. Here, we have described several ways to investigate the function of EVs during Plasmodium infection. For instance, the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PKH67 Green Fluorescent Cell Linker Mini Kit	Sigma-Aldrich	MINI67-1KT
Diluent C	Sigma-Aldrich	G8278
poly-L-lysine	Sigma-Aldrich	P8920
PBS	ThermoFisher - Gibco	10010023
Phalloidin CF594	Biotium	#00045
Hoechst 33342	ThermoFisher	H3570
ProLong Gold Antifade Mountant	ThermoFisher	P36934
Rhodamine B isothiocyanate–Dextran	ThermoFisher	R9379-250MG
Insert with PET membrane transparent Falcon for plate 24 wells	Falcon	353095
Endothelial Cell Growth Medium MV	Promocell	C-22020
Puromycin dihydrochloride	Sigma-Aldrich	P9620-10ML
MTS Cell Proliferation Colorimetric Assay Kit	Biovision	K300-500
hexadimethrine bromide	Sigma-Aldrich	107689-10G
MISSION Lenti microRNA, Human hsa-miR-451a	Sigma-Aldrich	HLMIR0583
MISSION Lenti microRNA, ath-miR416, Negative Control 1 Transduction Particles	Sigma-Aldrich	NCLMIR001
MISSION Lenti microRNA, Human	Sigma-Aldrich	NCLMIR0001
Leica TCS SP5	Leica Microsystems
miRNeasy mini Kit	Qiagen	217004
TaqMan MicroRNA Reverse Transcription Kit 1000 reactions	ThermoFisher	4366597
hsa-mir-451a RT/750 PCR rxns	ThermoFisher	001141
U6 snRNA	ThermoFisher	001973
TaqMan Universal Master Mix II, with UNG	ThermoFisher	4440038
StepOnePlus Real-Time PCR System	ThermoFisher	4376600