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      Extracellular vesicles from Echinococcus granulosus larval stage: Isolation, characterization and uptake by dendritic cells

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          Abstract

          The secretion of extracellular vesicles (EVs) in helminth parasites is a constitutive mechanism that promotes survival by improving their colonization and adaptation in the host tissue. In the present study, we analyzed the production of EVs from supernatants of cultures of Echinococcus granulosus protoscoleces and metacestodes and their interaction with dendritic cells, which have the ability to efficiently uptake and process microbial antigens, activating T lymphocytes. To experimentally increase the release of EVs, we used loperamide, a calcium channel blocker that increases the cytosolic calcium level in protoscoleces and EV secretion. An exosome-like enriched EV fraction isolated from the parasite culture medium was characterized by dynamic light scattering, transmission electron microscopy, proteomic analysis and immunoblot. This allowed identifying many proteins including: small EV markers such as TSG101, SDCBP, ALIX, tetraspanins and 14-3-3 proteins; proteins involved in vesicle-related transport; orthologs of mammalian proteins involved in the immune response, such as basigin, Bp29 and maspardin; and parasite antigens such as antigen 5, P29 and endophilin-1, which are of special interest due to their role in the parasite-host relationship. Finally, studies on the EVs-host cell interaction demonstrated that E. granulosus exosome-like vesicles were internalized by murine dendritic cells, inducing their maturation with increase of CD86 and with a slight down-regulation in the expression of MHCII molecules. These data suggest that E. granulosus EVs could interfere with the antigen presentation pathway of murine dendritic cells inducing immunoregulation in the host. Further studies are needed to better understand the role of these vesicles in parasite survival and as diagnostic markers and new vaccines.

          Author summary

          Human cystic echinococcosis, caused by chronic infection with the larval stage of Echinococcus granulosus, affects over 1 million people worldwide. This helminth parasite secretes numerous excretory/secretory products that are in contact with host tissues where it establishes hydatid cysts. In this study, we comprehensively characterized extracellular vesicles (EVs) from E. granulosus protoscoleces and metacestodes, and demonstrated for the first time that the exosome-like vesicles from helminths can interact with host dendritic cells and carry several immunoregulatory proteins. This study provides valuable data on cestode-host immune communication. Nevertheless, further research on EVs is needed to fully understand their role in the parasite-host interface and obtain new data concerning their function as therapeutic markers and diagnostic tools.

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          Most cited references64

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          Rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner.

          Multivesicular bodies (MVBs) are membranous structures within 60-100 nm diameter vesicles accumulate. MVBs are generated after invagination and pinching off of the endosomal membrane in the lumen of the vacuole. In certain cell types, fusion of MVBs with the plasma membrane results in the release of the internal vesicles called exosomes. In this report we have examined how an increase in cytosolic calcium affects the development of MVBs and exosome release in K562 cells overexpressing GFP-Rab11 wt or its mutants. In cells overexpressing the Rab11Q70 L mutant or Rab11 wt, an increase in the cytosolic calcium concentration induced by monensin caused a marked enlargement of the MVBs. This effect was abrogated by the membrane permeant calcium chelator BAPTA-AM. We also examined the behavior of MVBs in living cells by time lapse confocal microscopy. Many MVBs, decorated by wt or Q70L mutant GFP-Rab11, were docked and ready to fuse in the presence of a calcium chelator. This observation suggests that Rab11 is acting in the tethering/docking of MVBs to promote homotypic fusion, but that the final fusion reaction requires the presence of calcium. Additionally, a rise in intracellular calcium concentration enhanced exosome secretion in Rab11 wt overexpressing cells and reversed the inhibition of the mutants. The results suggest that both Rab11 and calcium are involved in the homotypic fusion of MVBs.
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            Autophagy and multivesicular bodies: two closely related partners.

            In the majority of cell types, multivesicular bodies (MVBs) are a special kind of late endosomes, crucial intermediates in the internalization of nutrients, ligands and receptors through the endolysosomal system. ESCRT-0, I, II and III (endosomal sorting complex required for transport) are involved in the sorting of proteins into MVBs, generating the intraluminal vesicles. Autophagy is a lysosomal degradation pathway for cytoplasmic components such as proteins and organelles. The autophagosome, a well-characterized structure of the autophagy pathway, can fuse with endocytic structures such as MVBs to generate the amphisome. Finally, the amphisome fuses with the lysosome to degrade the material wrapped inside. Currently, clear evidence suggests that efficient autophagic degradation requires functional MVBs. This review highlights the most recent advances in our understanding of the molecular machinery that participates in MVB biogenesis and regulates the interplay between autophagy and this organelle.
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              Immunomodulation by helminth parasites: defining mechanisms and mediators.

              Epidemiological and interventional human studies, as well as experiments in animal models, strongly indicate that helminth parasitic infections can confer protection from immune dysregulatory diseases such as allergy, autoimmunity and colitis. Here, we review the immunological pathways that helminths exploit to downregulate immune responses, both against bystander specificities such as allergens and against antigens from the parasites themselves. In particular, we focus on a highly informative laboratory system, the mouse intestinal nematode, Heligmosomoides polygyrus, as a tractable model of host-parasite interaction at the cellular and molecular levels. Analysis of the molecules released in vitro (as excretory-secretory products) and their cellular targets is identifying individual parasite molecules and gene families implicated in immunomodulation, and which hold potential for future human therapy of immunopathological conditions. Copyright © 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, CA USA )
                1935-2727
                1935-2735
                7 January 2019
                January 2019
                : 13
                : 1
                : e0007032
                Affiliations
                [1 ] Laboratorio de Zoonosis Parasitarias, Departamento de Biología, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata (UNMdP), Funes, Nivel Cero, Mar del Plata, Argentina
                [2 ] Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina
                [3 ] Departamento de Química, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata (UNMdP), Funes, Nivel 2, Mar del Plata, Argentina
                IRNASA, CSIC, SPAIN
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-0225-6363
                Article
                PNTD-D-18-00663
                10.1371/journal.pntd.0007032
                6344059
                30615613
                693a1b62-3d35-4a85-aa76-2ba0ecbd4774
                © 2019 Nicolao et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 24 April 2018
                : 28 November 2018
                Page count
                Figures: 3, Tables: 0, Pages: 24
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100003074, Agencia Nacional de Promoción Científica y Tecnológica;
                Award ID: PICT 2012 Nº2668
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100007070, Universidad Nacional de Mar del Plata;
                Award ID: Grant EXA 760/16
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100007070, Universidad Nacional de Mar del Plata;
                Award ID: Grant EXA 761/16
                Award Recipient :
                This work was supported by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT-Grant PICT 2012 Nº2668), CONICET (PIP 2015 N°11220150100406) and Universidad Nacional de Mar del Plata (Grant EXA 760/16 and EXA761/16), Argentina. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Vesicles
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Helminths
                Cestodes
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Flatworms
                Cestodes
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Immune Cells
                Antigen-Presenting Cells
                Dendritic Cells
                Biology and Life Sciences
                Immunology
                Immune Cells
                Antigen-Presenting Cells
                Dendritic Cells
                Medicine and Health Sciences
                Immunology
                Immune Cells
                Antigen-Presenting Cells
                Dendritic Cells
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Vesicles
                Exosomes
                Medicine and Health Sciences
                Parasitic Diseases
                Biology and Life Sciences
                Biochemistry
                Proteins
                Cytoskeletal Proteins
                Biology and Life Sciences
                Biochemistry
                Proteins
                Protein Domains
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Helminths
                Custom metadata
                vor-update-to-uncorrected-proof
                2019-01-23
                All relevant data are within the paper and its Supporting Information files.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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