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      Release of Luminal Exosomes Contributes to TLR4-Mediated Epithelial Antimicrobial Defense

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          Abstract

          Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. They are speculated to transfer molecules to neighboring or distant cells and modulate many physiological and pathological procedures. Exosomes released from the gastrointestinal epithelium to the basolateral side have been implicated in antigen presentation. Here, we report that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum. Release of exosomes involves activation of TLR4/IKK2 signaling through promoting the SNAP23-associated vesicular exocytotic process. Downregulation of let-7 family miRNAs by activation of TLR4 signaling increases SNAP23 expression, coordinating exosome release in response to C. parvum infection. Intriguingly, exosomes carry antimicrobial peptides of epithelial cell origin, including cathelicidin-37 and beta-defensin 2. Activation of TLR4 signaling enhances exosomal shuttle of epithelial antimicrobial peptides. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. Direct binding to the C. parvum sporozoite surface is required for the anti- C. parvum activity of released exosomes. Biliary epithelial cells also increase exosomal release and display exosome-associated anti- C. parvum activity following LPS stimulation. Our data indicate that TLR4 signaling regulates luminal exosome release and shuttling of antimicrobial peptides from the gastrointestinal epithelium, revealing a new arm of mucosal immunity relevant to antimicrobial defense.

          Author Summary

          Exosomes are secreted membranous nanovesicles produced by a variety of cells. Exosomes shuttle various molecules to transfer them to neighboring or distant cells, and have been implicated as mediators in cell-cell communications to modulate physiological and pathological procedures. Here, we report that luminal release of exosomal vesicles is an important component of Toll-like receptor 4 (TLR4)-associated gastrointestinal epithelial defense against infection by Cryptosporidium parvum, an obligate intracellular protozoan that infects gastrointestinal epithelial cells. Activation of TLR4 signaling in host epithelial cells following C. parvum infection promotes luminal release of epithelial exosomes and exosomal shuttling of antimicrobial peptides from the epithelium. By direct binding to the C. parvum surface, exosomal vesicles reveal anti- C. parvum activity. Activation of TLR4 signaling in epithelial cells after LPS stimulation also increases exosomal release and exosome-associated anti- C. parvum activity. Therefore, we speculate that TLR4-mediated exosome release may be relevant to innate mucosal immunity in general, representing a new target for therapeutic intervention for infectious diseases at the mucosal surface.

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          Most cited references 27

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          Membrane fusion: grappling with SNARE and SM proteins.

          The two universally required components of the intracellular membrane fusion machinery, SNARE and SM (Sec1/Munc18-like) proteins, play complementary roles in fusion. Vesicular and target membrane-localized SNARE proteins zipper up into an alpha-helical bundle that pulls the two membranes tightly together to exert the force required for fusion. SM proteins, shaped like clasps, bind to trans-SNARE complexes to direct their fusogenic action. Individual fusion reactions are executed by distinct combinations of SNARE and SM proteins to ensure specificity, and are controlled by regulators that embed the SM-SNARE fusion machinery into a physiological context. This regulation is spectacularly apparent in the exquisite speed and precision of synaptic exocytosis, where synaptotagmin (the calcium-ion sensor for fusion) cooperates with complexin (the clamp activator) to control the precisely timed release of neurotransmitters that initiates synaptic transmission and underlies brain function.
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            Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes.

            Association of major histocompatibility complex (MHC) class II molecules with peptides occurs in a series of endocytic vacuoles, termed MHC class II-enriched compartments (MIICs). Morphological criteria have defined several types of MIICs, including multivesicular MIICs, which are composed of 50-60-nm vesicles surrounded by a limiting membrane. Multivesicular MIICs can fuse with the plasma membrane, thereby releasing their internal vesicles into the extracellular space. The externalized vesicles, termed exosomes, carry MHC class II and can stimulate T-cells in vitro. In this study, we show that exosomes are enriched in the co-stimulatory molecule CD86 and in several tetraspan proteins, including CD37, CD53, CD63, CD81, and CD82. Interestingly, subcellular localization of these molecules revealed that they were concentrated on the internal membranes of multivesicular MIICs. In contrast to the tetraspans, other membrane proteins of MIICs, such as HLA-DM, Lamp-1, and Lamp-2, were mainly localized to the limiting membrane and were hardly detectable on the internal membranes of MIICs nor on exosomes. Because internal vesicles of multivesicular MIICs are thought to originate from inward budding of the limiting membrane, the differential distribution of membrane proteins on the internal and limiting membranes of MIICs has to be driven by active protein sorting.
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              Interferon modulation of cellular microRNAs as an antiviral mechanism.

              RNA interference through non-coding microRNAs (miRNAs) represents a vital component of the innate antiviral immune response in plants and invertebrate animals; however, a role for cellular miRNAs in the defence against viral infection in mammalian organisms has thus far remained elusive. Here we show that interferon beta (IFNbeta) rapidly modulates the expression of numerous cellular miRNAs, and that eight of these IFNbeta-induced miRNAs have sequence-predicted targets within the hepatitis C virus (HCV) genomic RNA. The introduction of synthetic miRNA-mimics corresponding to these IFNbeta-induced miRNAs reproduces the antiviral effects of IFNbeta on HCV replication and infection, whereas neutralization of these antiviral miRNAs with anti-miRNAs reduces the antiviral effects of IFNbeta against HCV. In addition, we demonstrate that IFNbeta treatment leads to a significant reduction in the expression of the liver-specific miR-122, an miRNA that has been previously shown to be essential for HCV replication. Therefore, our findings strongly support the notion that mammalian organisms too, through the interferon system, use cellular miRNAs to combat viral infections.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                April 2013
                April 2013
                4 April 2013
                : 9
                : 4
                Affiliations
                [1 ]Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, Nebraska, United States of America
                [2 ]Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota, United States of America
                [3 ]Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, Massachusetts, United States of America
                [4 ]Department of Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas, United States of America
                University of Virginia Health System, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: GH AYG ALR NDH XMC. Performed the experiments: GH AYG ALR BQH. Analyzed the data: GH AYG ALR NDH XMC. Contributed reagents/materials/analysis tools: HDW GZ NFL. Wrote the paper: GH XMC.

                Article
                PPATHOGENS-D-12-02699
                10.1371/journal.ppat.1003261
                3617097
                23592986

                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.

                Page count
                Pages: 13
                Funding
                This work was supported in part by grants from the National Institutes of Health (U01 AI095532) and the Tobacco Settlement Biomedical Research Program (CU LB692 and LB595) (to XMC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Immunology
                Immune Response
                Immunity
                Microbiology
                Emerging Infectious Diseases
                Host-Pathogen Interaction
                Parasitology
                Pathogenesis
                Molecular Cell Biology
                Nucleic Acids
                RNA
                Cellular Stress Responses
                Signal Transduction
                Medicine
                Gastroenterology and Hepatology
                Infectious Diseases
                Gastrointestinal Infections
                Parasitic Diseases

                Infectious disease & Microbiology

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