Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice
Rafael Prados-Rosales, … , Steven A. Porcelli, Arturo Casadevall
Rafael Prados-Rosales, … , Steven A. Porcelli, Arturo Casadevall
Published March 1, 2011
Citation Information: J Clin Invest. 2011;121(4):1471-1483. https://doi.org/10.1172/JCI44261.
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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.

Authors

Rafael Prados-Rosales, Andres Baena, Luis R. Martinez, Jose Luque-Garcia, Rainer Kalscheuer, Usha Veeraraghavan, Carmen Camara, Joshua D. Nosanchuk, Gurdyal S. Besra, Bing Chen, Juan Jimenez, Aharona Glatman-Freedman, William R. Jacobs Jr., Steven A. Porcelli, Arturo Casadevall

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Figure 5

Responses of macrophages to BCG MVs.

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Responses of macrophages to BCG MVs. Freshly isolated BMM and alveolar m...
Freshly isolated BMM and alveolar macrophage cell line (AMJ2-C8) were stimulated with MVs and with Pam3CSK4 in vitro (500,000 cells/500 μl of media). Supernatants and cell lysates were collected at time points ranging from 0 to 48 hours after stimulation (x axes of all graphs indicate time in hours). (A) The supernatants of cultures of WT (white bars) or TLR2-knockout (black bars) isolated BMMs were assayed for cytokines IL-1β, IL-10, IL-12p70, TNF-α, and chemo­kines CXCL1 by multiplex ELISA (MSD Systems). Cytokines IL-6 and MIP-1α were analyzed by standard ELISA. Data represent mean ± SEM. (B) The levels of Cox-2 and MMP-9 were analyzed by Western blot and zymography, respectively. For Cox-2 analysis (top), lysates were made from cells (AMJ2-C8 alveolar macrophage cell line (AM), WT BMM, or Tlr2–/– BMM collected at the indicated times, ranging from 0 to 48 hours after addition of PBS (unstimulated), Pam3CSK4, or BCG MVs. For measurement of MMP-9 activity, culture supernatants were collected at the indicated times following stimulation as shown. The graphs at the bottom show levels of MMP-9 activity relative to a recombinant MMP-9 protein standard. The data of the Cox-2 and MMP-9 analysis are representative of 3 independent experiments. Data represent mean ± SEM.