Name: evaluation of extracellular vesicle function during malaria infection
Uploaded: 2018-02-14T14:30:00
Description: Watch this Scientific Journal Video about Evaluation of Extracellular Vesicle Function During Malaria Infection at JoVE.com

This study was financially supported in part by the Novartis foundation for medical- and biological research (to PYM), the Gottfried and Julia Bangerter-Rhyner-Stiftung (to MW and PYM), and the research pool of the University of Fribourg (to PYM). Additional grants include the Swiss Government Excellence Scholarships for Foreign Scholars (to KAB and SM). We thank Isabelle Fellay and Solange Kharoubi Hess for technical support.

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
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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 ...

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 infection3. It is possible that they play two important roles: on one hand, they might contribute to the pathogenesis by activating macrophages4,5; and on the other hand, they might mediate cellular communication between parasites and between parasites and host6,7. In fact, parasites can transfer proteins or nucleic acids between each other via EVs. For example, Trypanosoma brucei rhodesiense EVs can transfer virulence factor Serum Resistance-Associated (SRA), and can target both other T. brucei and host erythrocytes8. Furthermore, P. falciparum-iRBCs communicate between each other by transferring nucleic acids within EVs. This allows the parasites to optimize and synchronize its growth. In fact, EVs might be the major regulator of gametocyte conversion, and therefore contribute to the regulation of the transmission stage7.
Not only do EVs regulate the parasites, they also mediate parasite-host interactions. We recently discovered that EVs from iRBCs contain host-derived microRNAs (miRNAs; small RNA species in the range of 21-25 nucleotides9) that were taken up by human endothelial cells. The miRNAs in the EVs form a stable complex with Ago2 (a member of the RNA-induced silencing complex), which once delivered to the recipient cells, is capable of specifically silencing gene expression and affecting the barrier properties of the cells10. Standard protocols have been developed to investigate the function of EVs. Here, we describe first a protocol that allows the fluorescent labeling of EVs to investigate their uptake by recipient cells. In addition, by using a confocal microscope, it is possible to track the EV&#39;s fate inside the cell. Several fluorescent dyes can be used to track EVs. The amine-reactive dye, 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) and Calcein-AM become fluorescent once inside the vesicles. We prefer to use the amphiphilic label, PKH, because it gives a brighter and more uniform signal. This approach provides important information to understand the interactions between EVs and recipient cells. While in some cases EVs bind to the surface of the cells, some vesicles are rapidly taken up. Upon uptake, EVs deliver their cargoes to the cells, in which they exert their regulatory functions.
Here, we describe a protocol to measure the barrier function of the endothelial cells in vitro by quantifying the transfer of a fluorescent dextran through a cellular monolayer. More sensitive tracers can be used such as radiolabeled markers. However, they require special safety precautions for use. Other assays exist to measure the in vitro barrier function such as transendothelial electrical resistance (TEER), which measures tight junction integrity. Finally visualizing ZO-1, a tight junction protein, by immunofluorescence allows assessment of tight junction integrity as well10. Since EVs are complex and heterogeneous entities containing several cargoes with potential regulatory characteristic, it is useful to overexpress a specific RNA to study its effect on the recipient cell. Therefore, we also define a protocol that aims to generate stable cell lines expressing the miRNA of interest10.

<table>
	<tbody>
		<tr>
			<td>PKH67 Green Fluorescent Cell Linker Mini Kit</td>
			<td>Sigma-Aldrich</td>
			<td>MINI67-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Diluent C</td>
			<td>Sigma-Aldrich</td>
			<td>G8278</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher - Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin CF594</td>
			<td>Biotium</td>
			<td>#00045</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>ThermoFisher</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine B isothiocyanate&#8211;Dextran</td>
			<td>ThermoFisher</td>
			<td>R9379-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Insert with PET membrane transparent Falcon&#160;for plate 24 wells</td>
			<td>Falcon</td>
			<td>353095</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth&#160;Medium MV</td>
			<td>Promocell</td>
			<td>C-22020</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9620-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>MTS Cell Proliferation Colorimetric Assay Kit</td>
			<td>Biovision</td>
			<td>K300-500</td>
			<td></td>
		</tr>
		<tr>
			<td>hexadimethrine bromide</td>
			<td>Sigma-Aldrich</td>
			<td>107689-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION&#160;Lenti microRNA, Human hsa-miR-451a</td>
			<td>Sigma-Aldrich</td>
			<td>HLMIR0583</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION Lenti microRNA, ath-miR416, Negative Control 1 Transduction Particles</td>
			<td>Sigma-Aldrich</td>
			<td>NCLMIR001</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION&#160;Lenti microRNA, Human</td>
			<td>Sigma-Aldrich</td>
			<td>NCLMIR0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP5</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy mini Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Reverse Transcription Kit 1000 reactions</td>
			<td>ThermoFisher</td>
			<td>4366597</td>
			<td></td>
		</tr>
		<tr>
			<td>hsa-mir-451a&#160;RT/750 PCR rxns</td>
			<td>ThermoFisher</td>
			<td>001141</td>
			<td></td>
		</tr>
		<tr>
			<td>U6 snRNA</td>
			<td>ThermoFisher</td>
			<td>001973</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal Master Mix II, with UNG</td>
			<td>ThermoFisher</td>
			<td>4440038</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOnePlus Real-Time PCR System</td>
			<td>ThermoFisher</td>
			<td>4376600</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of extracellular vesicle function during malaria infection

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.

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...

Watch this Scientific Journal Video about Evaluation of Extracellular Vesicle Function During Malaria Infection at JoVE.com

Evaluation of Extracellular Vesicle Function During Malaria Infection

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.

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

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