Name: Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus (Video) | JoVE Journal
Uploaded: 2025-03-28T13:15:00.000+00:00
Duration: 9 min 4 s
Description: Learn to isolate small extracellular vesicles from Echinococcus granulosus and analyze their uptake by dendritic cells. This method reveals immunomodulatory mechanisms in parasite-host interactions.

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5 minutes

The authors acknowledge Lic. Cecilia Guti&#233;rrez Ayesta (Servicio de Microscop&#237;a Electr&#243;nica, CONICET, Bah&#237;a Blanca, Argentina) and Lic. Leonardo Sechi and Lic. Eliana Maza (INIFTA, Universidad Nacional de La Plata, Argentina) for the technical assistance with transmission electron microscopy and dynamic light scattering, respectively. We also thank Dra. Graciela Salerno, Dra. Corina Ber&#243;n and Dr. Gonzalo Cal&#243; for the use of the ultracentrifuge at the INBIOTEC-CONICET-FIBA, Argentina. The authors gratefully acknowledge Lic. Kelly (SENASA, Mar del Plata, Argentina) and Lic. H. N&#250;nez Garc&#237;a (CONICET, Universidad Nacional de Mar del Plata, Argentina) for their collaboration in the welfare assessment of mice, and Med. Vet. J. Reyno, Med. Vet. S. Gonzalez, and Med. Vet L. Netti for their contribution to obtaining parasite material. This work, including the costs of experimentation, reagents, and equipment, was supported by the PICT 2020 No. 1651 financed by the ANPCyT.

The authors declare that they have no conflicts of interest.

The protocol workflow for culturing parasites, isolating parasite-derived sEVs, differentiating dendritic cells from bone marrow, and analyzing sEV uptake by these cells is outlined in Figure 1. The aim was to describe in detail each protocol section that may be carried out as a whole or separately, highlighting the major considerations to guarantee the implementation of the methodology. The analysis of the population of EVs obtained from complete parasitic organisms has a concrete impact on...

Echinococcus granulosus is a zoonotic parasitic helminth responsible for a long-term infection known as cystic echinococcosis1. In intermediate hosts, such as livestock and humans, the parasite infection primarily affects the liver and lungs, where the larval stage develops as fluid-filled cysts or metacestodes containing protoscoleces (a larva itself). Like all cestodes, this parasite lacks both digestive and excretory systems and has, therefore, evolved active endocytic and exocytic cellular processes to regulate the uptake and excretion of metabolites as well as the release of extracellular vesicles2,3. Extracellular vesicles (EVs) are lipid bilayer-enclosed particles secreted by apparently all cell types. In particular, small extracellular vesicles (sEVs), defined as EVs smaller than 200 nm regardless of their biogenesis origin4, can act as intercellular immune mediators. This function is especially significant in parasites, which rely on host immunomodulation to ensure their survival3. Immune manipulation is achieved through the uptake of sEVs by host dendritic cells, the only cells capable of activating naive T cells in vivo and initiating an adaptive immune response that will lead to chronic infection by these parasitic worms. Dendritic cells, professional antigen-presenting cells of the innate immune system, process and load antigenic peptides onto Major Histocompatibility Complex Class I and Class II (MHC I and MHC II) and exhibit them on their membranes for exclusive na&#239;ve T cell priming (CD8+ and CD4+ T cells, respectively)5. Following dendritic cells induce their maturation by induction of expression of the co-stimulatory markers CD80/CD86 and CD40 and MHC-II and migrate from peripheral tissues to secondary lymphoid organs upon recognizing foreign antigens, loading them for exclusive na&#239;ve T cell priming6. Thus, the overall goal of this protocol is to study the helminth parasite-host communication in a realistic manner, analyzing the packaging and delivery of parasitic components in the form of sEVs, which, upon reaching the host immune cells, influence the development of infection and the progression of the chronic parasitic disease.
Addressing the analysis of the helminth-host interface through the study of sEVs has several advantages. First, the tegument, the outer covering of flatworms, is a double membrane structure that constitutes a major crossing point between the parasite and its host, allowing sEVs to be readily generated or permeated from this structure7. Second, sEVs are highly loaded with protein antigens from all stages of the parasite life cycle, representing the natural way through which the host immune system samples antigens during worm infection8,9. Due to their biological production, ease of purification (without requiring tissue disruption or protein fractionation), and direct interaction with host cells, helminth sEVs enable the development of in vitro experiments to simulate the in vivo conditions of parasite-host interaction. Finally, sEVs represent the possibility of having parasitic structures that can be phagocytosed or internalized by host cells, overcoming the impossibility of doing so with whole parasites, particularly in cases of encysted worms.
Considering the advantages mentioned and the fact that helminthiases are prevalent and typically chronic diseases in which parasites presumably manipulate the host immune system as a survival strategy, the isolation of parasite-derived EVs and their study in interaction with dendritic cells provides a valuable framework to explore this immunomodulation10. In this sense, it has been described that the internalization of EVs from helminths, including nematodes and platyhelminths such as Schistosoma mansoni, Fasciola hepatica, Brugia malayi, and E. granulosus, induces the maturation and activation of dendritic cells9,11,12,13,14,15.
The isolation of helminth-derived EVs not only enables the study of immunological interactions, potentially leading to the development of protective vaccines or immunotherapeutic agents for allergic or autoimmune diseases, but also facilitates the exploration of other biological interactions and functions8,16,17. In this context, EVs, which play a role in the natural history of parasitic infections, could be utilized to investigate parasite development and interactions with specific host cells. Moreover, they could have potential applications as early or differential biomarkers for the diagnosis of parasitic diseases, monitoring therapeutic responses, and contributing to the control and management of parasitic infections17,18.
In addition, as previously demonstrated, the larval stage of E. granulosus is susceptible to changes in cytosolic calcium concentration, which, besides playing a role in parasite viability, also controls the exocytosis rate19,20. In this context, and knowing that intracellular calcium elevation enhances EV release, using an intracellular calcium enhancer as loperamide could be a crucial strategy to increase the number of EVs. This approach is particularly interesting for cellular systems that require large populations to generate an adequate quantity of EVs for cargo and functional analysis11,21,22. The current protocol (Figure 1) details the methods for obtaining pure cultures of E. granulosus larval stage and the conditions that enhance sEV production. It also describes the workflow for the isolation and characterization of these vesicles, as well as their uptake by murine dendritic cells, an essential step in the initial study of host immune system modulation.

<table><tbody><tr><td>1.5 mL tubes</td><td>Henso</td><td>N14059</td><td></td></tr><tr><td>24-well plate&amp;nbsp;</td><td>JetBiofil</td><td>CAP011024</td><td>Polystyrene, flat bottom, Sterile</td></tr><tr><td>6-well plate&amp;nbsp;&amp;nbsp;</td><td>Henso Medical Co. Ltd.</td><td>N14221</td><td>Flat-shape bottom, PS material, Sterile</td></tr><tr><td>70 mm polypropylene cell strainer</td><td>Biologix Group Limited</td><td>15-1070</td><td>Sterile</td></tr><tr><td>Alcian blue 8 GX dye&amp;nbsp;</td><td>Santa Cruz</td><td>sc-214517B</td><td></td></tr><tr><td>Automatic CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Nuarire</td><td>UN-5100E/G DH</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Wiener lab</td><td>1443151</td><td></td></tr><tr><td>CD11c Monoclonal antibody-PECy5&amp;nbsp;&amp;nbsp; 100 &amp;micro;g</td><td>eBioscience</td><td>15-0114-82</td><td>clone (N418)</td></tr><tr><td>CD40 Monoclonal antibody-FITC 100 &amp;micro;g</td><td>eBioscience</td><td>11-0402-82</td><td>clone (HM40-3)</td></tr><tr><td>CD80 Monoclonal antibody-APC 100 &amp;micro;g</td><td>eBioscience</td><td>17-0801-82</td><td>clone (16-10A-1)</td></tr><tr><td>CD86 Monoclonal antibody-FITC&amp;nbsp; 100 &amp;micro;g</td><td>eBioscience</td><td>11-0862-82</td><td>clone (GL-1)</td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>IEC CL31R Multispeed</td><td></td></tr><tr><td>Confocal Microscope</td><td>Nikon</td><td>Nikon Confocal Microscope C1</td><td></td></tr><tr><td>Conical tubes 15 mL dia.17 x 120 mm</td><td>Citotest</td><td>4610-1865</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>107K4034</td><td></td></tr><tr><td>D-Glucose</td><td>Merk</td><td>1.78343</td><td></td></tr><tr><td>Dimethyl Sulfoxide&amp;nbsp;</td><td>Anedra</td><td>6646</td><td></td></tr><tr><td>Fetal Bovine Serum 500 mL</td><td>Sigma-Aldrich&amp;nbsp;&amp;nbsp;</td><td>12352207</td><td></td></tr><tr><td>Flow Cytometry System</td><td>BD Biosciences</td><td>BD FACSCanto&amp;trade; II</td><td></td></tr><tr><td>Folded Capillary Zeta Cell&amp;nbsp;</td><td>Malvern Panalytical</td><td>DTS1070</td><td></td></tr><tr><td>Gentamicin sulfate salt</td><td>Sigma</td><td>G1264</td><td></td></tr><tr><td>Glutaraldehyde solution</td><td>Fluka</td><td>49630</td><td></td></tr><tr><td>Hepes</td><td>Gibco</td><td>11344041</td><td></td></tr><tr><td>Hypodermic&amp;nbsp; needle 21 G x 1&quot;25/8</td><td>Weigao</td><td></td><td>Sterile</td></tr><tr><td>Hypodermic&amp;nbsp; needle 25 G x 5/8&quot;</td><td>Weigao</td><td></td><td>Sterile</td></tr><tr><td>Inverted microscope&amp;nbsp;</td><td>Leica</td><td>DMIL LED Fluo&amp;nbsp;</td><td></td></tr><tr><td>Ketamine</td><td>Holliday</td><td></td><td></td></tr><tr><td>Lipopolysaccharide&amp;nbsp; 5 mg</td><td>Invitrogen</td><td>tlrl-rslps</td><td>LPS from the Gram-negative bacteria E. coli K12 . TLR2/4 Agonists</td></tr><tr><td>Loperamide hydrochloride</td><td>Sigma-Aldrich</td><td>5.08162</td><td></td></tr><tr><td>Medium 199&amp;nbsp;</td><td>Gibco</td><td>11150059</td><td></td></tr><tr><td>Methylene Blue</td><td>Anedra</td><td>6337</td><td></td></tr><tr><td>MHC class I (H2kb) Monoclonal antibody-PE 100 &amp;micro;g</td><td>eBioscience</td><td>12-5958-82</td><td>clone (AF6-88.5.5.3)</td></tr><tr><td>MHC class II (IA/IE) Monoclonal antibody-FITC&amp;nbsp; 100 &amp;micro;g</td><td>eBioscience</td><td>11-5321-82</td><td>clone (M5/114.15.2)</td></tr><tr><td>Microscope</td><td>Olympus</td><td>CX31</td><td></td></tr><tr><td>Mouse recombinant murine Flt3L.</td><td>PrepoTech</td><td>250-31L-10UG</td><td></td></tr><tr><td>Nanodrop</td><td>Thermo Scientific</td><td>ND-One</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Agar Scientific</td><td>R1018</td><td></td></tr><tr><td>Penicillin G sodium salt</td><td>Sigma</td><td>P3032-10MU</td><td></td></tr><tr><td>PKH26</td><td>Sigma-Aldrich</td><td>MINI26</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Timper</td><td></td><td>For Phosphate Buffered Saline (PBS)</td></tr><tr><td>RBC lysis buffer 100 mL</td><td>Roche</td><td>11814389001</td><td></td></tr><tr><td>RPMI medium&amp;nbsp; 1640 1x 500 mL</td><td>Sigma-Aldrich (Gibco)</td><td>11875093</td><td></td></tr><tr><td>Sodium Cacodylate&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C0250</td><td></td></tr><tr><td>Sodium Chloride</td><td>Anedra</td><td>7647-14-5</td><td>For Phosphate Buffered Saline (PBS)</td></tr><tr><td>Sodium Phosphate Dibasic (Anhydrous) p. a.</td><td>Biopack</td><td>1639.07</td><td>For Phosphate Buffered Saline (PBS)</td></tr><tr><td>Streptomycin sulfate salt</td><td>Sigma</td><td>S9137</td><td></td></tr><tr><td>Syringe 10 mL</td><td>Bremen</td><td></td><td>Sterile</td></tr><tr><td>Thickwall polycarbonate tubes</td><td>Beckman Coulter</td><td></td><td>13 x 55 mm , nominal capacity 4 mL</td></tr><tr><td>Transmission Electron Microscope</td><td>Jeol</td><td>JEOL JSM 100CX II</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman</td><td>Optima LE-80k&amp;nbsp;</td><td>90 Ti rotor</td></tr><tr><td>Xylazine</td><td>Richmond</td><td></td><td></td></tr><tr><td>Zetasizer Nano&amp;nbsp;</td><td>Malvern&amp;nbsp;</td><td>Nano ZSizer-ZEN3600</td><td>To perform Dynamic Light scattering and zeta potential measurements</td></tr></tbody></table>

isolation and dendritic cell-uptake of small extracellular vesicles from echinococcus granulosus

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

A flowchart summarizing the main steps for maintaining pure cultures of the E. granulosus larval stage, the isolation and characterization of sEVs, and their uptake by murine dendritic cells are shown in Figure 1. To achieve high sEV production from E. granulosus protoscoleces and metacestodes, an in vitro culture method previously developed in the laboratory was employed to maximize the survival and metabolic homeostasis of the studied parasite (<strong class="xfi...

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

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Learn to isolate small extracellular vesicles from Echinococcus granulosus and analyze their uptake by dendritic cells. This method reveals immunomodulatory mechanisms in parasite-host interactions.

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

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