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      Immunomodulatory Impact of Leishmania-Induced Macrophage Exosomes: A Comparative Proteomic and Functional Analysis

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      PLoS Neglected Tropical Diseases

      Public Library of Science

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          Abstract

          Released by many eukaryotic cells, the exosomes are 40–100 nm vesicles shown to operate over the complex processes of cell-cell communication. Among the metazoan cell lineages known to generate exosomes is the mononuclear phagocyte lineage, a lineage that parasites such as Leishmania are known to subvert as host cells. We previously reported that mouse macrophage signaling and functions are modified once co-incubated with exoproteome of Leishmania promastigotes. Using mass spectrometry analysis, we were curious to further compare the content of purified exosomes released by the J774 mouse macrophage cell line exposed or not to either LPS or to stationary phase Leishmania mexicana promastigotes. Collectively, our analyses resulted in detection of 248 proteins, ∼50–80% of which were shared among the three sources studied. Using exponentially modified protein abundance index (emPAI) and network analyses, we found that the macrophage exosomes display unique signatures with respect to composition and abundance of many functional groups of proteins, such as plasma membrane-associated proteins, chaperones and metabolic enzymes. Moreover, for the first time, L. mexicana surface protease GP63 is shown to be present in exosomes released from J774 macrophages exposed to stationary phase promastigotes. We observed that macrophage exosomes are able to induce signaling molecules and transcription factors in naive macrophages. Finally, using qRT-PCR, we monitored modulation of expression of multiple immune-related genes within macrophages exposed to exosomes. We found all three groups of exosomes to induce expression of immune-related genes, the ones collected from macrophages exposed to L. mexicana sharing properties with exosomes collected from macrophage left unexposed to any agonist. Overall, our results allowed depicting that protein sorting into macrophage-derived exosomes depends upon the cell status and how such distinct protein sorting can in turn impact the functions of naive J774 cells.

          Author Summary

          Secreted vesicles, such as exosomes, are now considered as an important route of communication among eukaryotic cells. Depending on the donor cell source and protein content, these vesicles are expected to distinctly impact the recipient cell properties. Here, three groups of exosomes released by the mouse macrophage cell line J774 exposed or not - naive exosomes - to either Leishmania mexicana promastigotes or to LPS were compared through proteomic analysis. Also, their biological activities on naive J774 macrophages were tested. Regardless of the source, the three groups of exosomes shared 50–80% of their proteins, although their relative abundances differed, especially those associated with the plasma membrane. Post exposure to one out of the three groups of exosomes, naive J774 recipient macrophages were compared for their profile of immune transcripts. Of note, whether they were exposed to either naive exosomes or to L. mexicana- induced exosomes, the naive J774 macrophages shared similar immune transcriptional signatures, the latter being distinct from the ones displayed by the macrophages exposed to LPS-induced exosomes. These data are discussed within the context of the unique cross talk that accounts for the early establishment of an immunomodulatory parasite such as Leishmania in its mammalian host.

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          Most cited references 28

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          Exosomes released from macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro and in vivo.

          Intracellular pathogens and the molecules they express have limited contact with the immune system. Here, we show that macrophages infected with intracellular pathogens Mycobacterium tuberculosis, M bovis BCG, Salmonella typhimurium, or Toxoplasma gondii release from cells small vesicles known as exosomes which contain pathogen-associated molecular patterns (PAMPs). These exosomes, when exposed to uninfected macrophages, stimulate a proinflammatory response in a Toll-like receptor- and myeloid differentiation factor 88-dependent manner. Further, exosomes isolated from the bronchoalveolar lavage fluid (BALF) of M bovis BCG-infected mice contain the mycobacteria components lipoarabinomannan and the 19-kDa lipoprotein and can stimulate TNF-alpha production in naive macrophages. Moreover, exosomes isolated from M bovis BCG- and M tuberculosis-infected macrophages, when injected intranasally into mice, stimulate TNF-alpha and IL-12 production as well as neutrophil and macrophage recruitment in the lung. These studies identify a previously unknown function for exosomes in promoting intercellular communication during an immune response to intracellular pathogens, and we hypothesize that extracellular release of exosomes containing PAMPs is an important mechanism of immune surveillance.
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            Exosomes released from infected macrophages contain Mycobacterium avium glycopeptidolipids and are proinflammatory.

            Mycobacterium avium is a major opportunistic pathogen in HIV-positive individuals and is responsible for increased morbidity and mortality in AIDS patients. M. avium express glycopeptidolipids (GPLs) as a major cell wall constituent, and recent studies suggest that GPLs play an important role in M. avium pathogenesis. In the present study we show that M. avium-infected macrophages release GPLs, which are trafficked from the phagosome through the endocytic network to multivesicular bodies. Prior studies have shown that multivesicular bodies can fuse with the plasma membrane releasing small 50 to 100 nm vesicles known as exosomes. We found that M. avium-infected macrophages release exosomes containing GPLs leading to the transfer of GPLs from infected to uninfected macrophages. Interestingly, exosomes isolated from M. avium-infected but not from uninfected macrophages can stimulate a proinflammatory response in resting macrophages. This proinflammatory response is dependent on Toll like receptor (TLR) 2, TLR4, and MyD88 suggesting that released exosomes contain M. avium-expressed TLR ligands. Our studies are the first to demonstrate that exosomes isolated from mycobacteria-infected macrophages can induce a proinflammatory response, and we hypothesize that exosomes play an important role in immune surveillance during intracellular bacteria infections.
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              Fast-response proteomics by accelerated in-gel digestion of proteins.

              Kinetics of in-gel digestion of proteins by modified and native trypsins was studied by MALDI TOF mass spectrometry using 18O-labeled peptides as internal standards. The effect of the temperature, enzyme concentration, digestion time, and surface area of gel pieces on the yield of digestion products was characterized. Based on the kinetic data, we developed a protocol that enabled the identification of gel-separated proteins with 30-min digestion time without compromising the peptide yield and the sensitivity compared to conventional protocols that typically rely upon overnight enzymatic cleavage. The accelerated digestion protocol was tested in identification of more than 120 proteins from budding and fission yeasts at the subpicomole level.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Negl Trop Dis
                PLoS Negl Trop Dis
                plos
                plosntds
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                1935-2727
                1935-2735
                May 2013
                2 May 2013
                : 7
                : 5
                Affiliations
                Departments of Microbiology & Immunology and Medicine, The Research Institute of the McGill University Health Centre, McGill University, Montréal, Québec, Canada
                Institut Pasteur, France
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: KH MO. Performed the experiments: KH. Analyzed the data: KH MO. Wrote the paper: KH MO.

                Article
                PNTD-D-12-01513
                10.1371/journal.pntd.0002185
                3642089
                23658846
                ea749622-c08f-42d7-b3ce-7f1568eec750

                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: 17
                Funding
                This study was supported by Canadian Institutes of Health Research grant no. MOP-12671 to MO ( www.cihr-irsc.gc.ca). Research in MO laboratory is supported by operating grants from the Canadian Institute of Health Research (CIHR), Fonds de Recherche en Santé du Québec (FRSQ) and Fonds de Recherche du Québec - Nature et Technologies (FQRNT). KH is the recipient of studentships from the Research Institute of the McGill University Health Centre (MUHC) and Centre for Host-Parasite Interactions (CHPI). 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 Cells
                Monocytes
                Immune Response
                Immunomodulation
                Microbiology
                Protozoology
                Parastic Protozoans
                Leishmania
                Parasitology
                Molecular Cell Biology
                Membranes and Sorting
                Signal Transduction
                Proteomics
                Protein Abundance
                Protein Interactions
                Medicine
                Infectious Diseases
                Neglected Tropical Diseases
                Leishmaniasis
                Parasitic Diseases
                Leishmaniasis

                Infectious disease & Microbiology

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