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      Profiling extracellular vesicle release by the filarial nematode Brugia malayi reveals sex-specific differences in cargo and a sensitivity to ivermectin

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          Abstract

          The filarial nematode Brugia malayi is an etiological agent of Lymphatic Filariasis. The capability of B. malayi and other parasitic nematodes to modulate host biology is recognized but the mechanisms by which such manipulation occurs are obscure. An emerging paradigm is the release of parasite-derived extracellular vesicles (EV) containing bioactive proteins and small RNA species that allow secretion of parasite effector molecules and their potential trafficking to host tissues. We have previously described EV release from the infectious L3 stage B. malayi and here we profile vesicle release across all intra-mammalian life cycle stages (microfilariae, L3, L4, adult male and female worms). Nanoparticle Tracking Analysis was used to quantify and size EVs revealing discrete vesicle populations and indicating a secretory process that is conserved across the life cycle. Brugia EVs are internalized by murine macrophages with no preference for life stage suggesting a uniform mechanism for effector molecule trafficking. Further, the use of chemical uptake inhibitors suggests all life stage EVs are internalized by phagocytosis. Proteomic profiling of adult male and female EVs using nano-scale LC-MS/MS described quantitative and qualitative differences in the adult EV proteome, helping define the biogenesis of Brugia EVs and revealing sexual dimorphic characteristics in immunomodulatory cargo. Finally, ivermectin was found to rapidly inhibit EV release by all Brugia life stages. Further this drug effect was also observed in the related filarial nematode, the canine heartworm Dirofilaria immitis but not in an ivermectin-unresponsive field isolate of that parasite, highlighting a potential mechanism of action for this drug and suggesting new screening platforms for anti-filarial drug development.

          Author summary

          Brugia malayi is a parasitic nematode and etiological agent of Lymphatic Filariasis (LF), a mosquito-borne Neglected Tropical Disease affecting approximately 120 million people globally. Brugia and other parasitic nematodes have the ability to modulate host biology and evade the immune response but the mechanisms by which they do this are unclear. One possibility is via immunomodulatory proteins and small RNAs packaged and released in extracellular vesicles (EV) that target host cells. Here we show that all Brugia life stages relevant to human infection release EVs that are internalized by murine macrophages. These EVs contain immunomodulatory proteins and show sex-specific differences in cargo providing insight into how B. malayi establishes and maintains infection. Critically, we show ivermectin, a drug used to treat LF and related diseases affects EV release, which provides new insight into how these drugs work and identifies a new screening paradigm to help identify future anti-parasitic drugs.

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          Most cited references 83

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          Extracellular vesicles: Exosomes, microvesicles, and friends

          Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for transfer between cells of membrane and cytosolic proteins, lipids, and RNA. Deficiencies in our knowledge of the molecular mechanisms for EV formation and lack of methods to interfere with the packaging of cargo or with vesicle release, however, still hamper identification of their physiological relevance in vivo. In this review, we focus on the characterization of EVs and on currently proposed mechanisms for their formation, targeting, and function.
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            Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.

            In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions. Only recently, scientists acknowledged the difficulty of separating exosomes from other types of extracellular vesicles, which precludes a clear attribution of a particular function to the different types of secreted vesicles. To shed light into this complex but expanding field of science, this review focuses on the definition of exosomes and other secreted extracellular vesicles. Their biogenesis, their secretion, and their subsequent fate are discussed, as their functions rely on these important processes.
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              B lymphocytes secrete antigen-presenting vesicles

              Antigen-presenting cells contain a specialized late endocytic compartment, MIIC (major histocompatibility complex [MHC] class II- enriched compartment), that harbors newly synthesized MHC class II molecules in transit to the plasma membrane. MIICs have a limiting membrane enclosing characteristic internal membrane vesicles. Both the limiting membrane and the internal vesicles contain MHC class II. In this study on B lymphoblastoid cells, we demonstrate by immunoelectron microscopy that the limiting membrane of MIICs can fuse directly with the plasma membrane, resulting in release from the cells of internal MHC class II-containing vesicles. These secreted vesicles, named exosomes, were isolated from the cell culture media by differential centrifugation followed by flotation on sucrose density gradients. The overall surface protein composition of exosomes differed significantly from that of the plasma membrane. Exosome-bound MHC class II was in a compact, peptide-bound conformation. Metabolically labeled MHC class II was released into the extracellular medium with relatively slow kinetics, 10 +/- 4% in 24 h, indicating that direct fusion of MIICs with the plasma membrane is not the major pathway by which MHC class II reaches the plasma membrane. Exosomes derived from both human and murine B lymphocytes induced antigen-specific MHC class II-restricted T cell responses. These data suggest a role for exosomes in antigen presentation in vivo.
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                Author and article information

                Contributors
                Role: Data curationRole: InvestigationRole: Writing – original draft
                Role: Data curationRole: InvestigationRole: Writing – review & editing
                Role: Investigation
                Role: ConceptualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: 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
                16 April 2018
                April 2018
                : 12
                : 4
                Affiliations
                [1 ] Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, Iowa, United States of America
                [2 ] Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, United States of America
                McGill University, CANADA
                Author notes

                The authors have declared that no competing interests exist.

                Article
                PNTD-D-18-00030
                10.1371/journal.pntd.0006438
                5919703
                29659599
                © 2018 Harischandra 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.

                Counts
                Figures: 8, Tables: 1, Pages: 25
                Product
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000060, National Institute of Allergy and Infectious Diseases;
                Award ID: AI117204
                Award Recipient :
                This work was funded by an R21 award (AI117204) to MJK and a K22 award (AI125473) to MZ by the National Institute of Allergy and Infectious Diseases ( www.niaid.nih.gov). 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
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Nematoda
                Brugia
                Brugia Malayi
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Vesicles
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Invertebrates
                Nematoda
                Brugia
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Blood Cells
                White Blood Cells
                Macrophages
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Immune Cells
                White Blood Cells
                Macrophages
                Biology and Life Sciences
                Immunology
                Immune Cells
                White Blood Cells
                Macrophages
                Medicine and Health Sciences
                Immunology
                Immune Cells
                White Blood Cells
                Macrophages
                Biology and Life Sciences
                Computational Biology
                Genome Analysis
                Gene Ontologies
                Biology and Life Sciences
                Genetics
                Genomics
                Genome Analysis
                Gene Ontologies
                Biology and Life Sciences
                Biochemistry
                Proteins
                Proteomes
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Vesicles
                Exosomes
                Medicine and Health Sciences
                Parasitic Diseases
                Custom metadata
                vor-update-to-uncorrected-proof
                2018-04-26
                All relevant data are within the paper and its supporting information files.

                Infectious disease & Microbiology

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