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Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice.

ABSTRACT: Bacteria naturally release membrane vesicles (MVs) under a variety of growth environments. Their production is associated with virulence due to their capacity to concentrate toxins and immunomodulatory molecules. In this report, we show that the 2 medically important species of mycobacteria, Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin, release MVs when growing in both liquid culture and within murine phagocytic cells in vitro and in vivo. We documented MV production in a variety of virulent and nonvirulent mycobacterial species, indicating that release of MVs is a property conserved among mycobacterial species. Extensive proteomic analysis revealed that only MVs from the virulent strains contained TLR2 lipoprotein agonists. The interaction of MVs with macrophages isolated from mice stimulated the release of cytokines and chemokines in a TLR2-dependent fashion, and infusion of MVs into mouse lungs elicited a florid inflammatory response in WT but not TLR2-deficient mice. When MVs were administered to mice before M. tuberculosis pulmonary infection, an accelerated local inflammatory response with increased bacterial replication was seen in the lungs and spleens. Our results provide strong evidence that actively released mycobacterial vesicles are a delivery mechanism for immunologically active molecules that contribute to mycobacterial virulence. These findings may open up new horizons for understanding the pathogenesis of tuberculosis and developing vaccines.

SUBMITTER: Prados-Rosales R

PROVIDER: S-EPMC3069770 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice.

Prados-Rosales Rafael R Baena Andres A Martinez Luis R LR Luque-Garcia Jose J Kalscheuer Rainer R Veeraraghavan Usha U Camara Carmen C Nosanchuk Joshua D JD Besra Gurdyal S GS Chen Bing B Jimenez Juan J Glatman-Freedman Aharona A Jacobs William R WR Porcelli Steven A SA Casadevall Arturo A

The Journal of clinical investigation 20110401 4

Bacteria naturally release membrane vesicles (MVs) under a variety of growth environments. Their production is associated with virulence due to their capacity to concentrate toxins and immunomodulatory molecules. In this report, we show that the 2 medically important species of mycobacteria, Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin, release MVs when growing in both liquid culture and within murine phagocytic cells in vitro and in vivo. We documented MV productio ...[more]

PMID: 21364279

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Project description:Oral delivery of Gram positive bacteria, often derived from the genera Lactobacillus or Bifidobacterium, can modulate immune function. Although the exact mechanisms remain unclear, immunomodulatory effects may be elicited through the direct interaction of these bacteria with the intestinal epithelium or resident dendritic cell (DC) populations. We analyzed the immune activation properties of Lactobacilli and Bifidobacterium species and made the surprising observation that cellular responses in vitro were differentially influenced by the presence of serum, specifically the extracellular vesicle (EV) fraction. In contrast to the tested Lactobacilli species, tested Bifidobacterium species induce TLR2/6 activity which is inhibited by the presence of EVs. Using specific TLR ligands, EVs were found to enhance cellular TLR2/1 and TLR4 responses while TLR2/6 responses were suppressed. No effect could be observed on cellular TLR5 responses. We determined that EVs play a role in bacterial aggregation, suggesting that EVs interact with bacterial surfaces. EVs were found to slightly enhance DC phagocytosis of Bifidobacterium breve whereas phagocytosis of Lactobacillus rhamnosus was virtually absent upon serum EV depletion. DC uptake of a non-microbial substance (dextran) was not affected by the different serum fractions suggesting that EVs do not interfere with DC phagocytic capacity but rather modify the DC-microbe interaction. Depending on the microbe, combined effects of EVs on TLR activity and phagocytosis result in a differential proinflammatory DC cytokine release. Overall, these data suggest that EVs play a yet unrecognized role in host-microbe responses, not by interfering in recipient cellular responses but via attachment to, or scavenging of, microbe-associated molecular patterns. EVs can be found in any tissue or bodily fluid, therefore insights into EV-microbe interactions are important in understanding the mechanism of action of potential probiotics and gut immune homeostasis.

Project description:Extracellular vesicles are membranous structures shed by almost every living cell. Bacterial gram-negative outer membrane vesicles (OMVs) and gram-positive membrane vesicles (MVs) play important roles in adaptation to the surrounding environment, cellular components' exchange, transfer of antigens and virulence factors, and infection propagation. Streptococcus pneumoniae is considered one of the priority pathogens, with a global health impact due to the increase in infection burden and growing antibiotic resistance. We isolated MVs produced from the S. pneumoniae reference strain (R6) and purified them via size exclusion chromatography (SEC) to remove soluble protein impurities. We characterized the isolated MVs by nanoparticle tracking analysis (NTA) and measured their particle size distribution and concentration. Isolated MVs showed a mean particle size range of 130-160 nm and a particle yield of around 1012 particles per milliliter. Cryogenic transmission electron microscopy (cryo-TEM) images revealed a very heterogeneous nature of isolated MVs with a broad size range and various morphologies, arrangements, and contents. We incubated streptococcal MVs with several mammalian somatic cells, namely, human lung epithelial A549 and human keratinocytes HaCaT cell lines, and immune cells including differentiated macrophage-like dTHP-1 and murine dendritic DC2.4 cell lines. All cell lines displayed excellent viability profile and negligible cytotoxicity after 24-h incubation with MVs at concentrations reaching 106 MVs per cell (somatic cells) and 105 MVs per cell (immune cells). We evaluated the uptake of fluorescently labeled MVs into these four cell lines, using flow cytometry and confocal microscopy. Dendritic cells demonstrated prompt uptake after 30-min incubation, whereas other cell lines showed increasing uptake after 2-h incubation and almost complete colocalization/internalization of MVs after only 4-h incubation. We assessed the influence of streptococcal MVs on antigen-presenting cells, e.g., dendritic cells, using enzyme-linked immunosorbent assay (ELISA) and observed enhanced release of tumor necrosis factor (TNF)-?, a slight increase of interleukin (IL)-10 secretion, and no detectable effect on IL-12. Our study provides a better understanding of gram-positive streptococcal MVs and shows their potential to elicit a protective immune response. Therefore, they could offer an innovative avenue for safe and effective cell-free vaccination against pneumococcal infections.

Dataset Information

Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice.

Publications

Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice.

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