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Membrane Vesicles Are Immunogenic Facsimiles of Salmonella typhimurium That Potently Activate Dendritic Cells, Prime B and T Cell Responses, and Stimulate Protective Immunity In Vivo¹

Robert C. Alaniz^*, Brooke L. Deatherage, Jimmie C. Lara and Brad T. Cookson^2,*,

Departments of ^* Laboratory Medicine and Microbiology, University of Washington, Seattle, WA 98195

Gram-negative bacteria produce membrane vesicles (MVs) from their outer membrane during growth, although the mechanism for MV production and the advantage that MVs provide for bacterial survival in vivo remain unknown. MVs function as an alternate secretion pathway for Gram-negative bacteria; therefore, MV production in vivo may be one method by which bacteria interact with eukaryotic cells. However, the interactions between MVs and cells of the innate and adaptive immune systems have not been studied extensively. In this study, we demonstrate that MVs from Salmonella typhimurium potently stimulated professional APCs in vitro. Similar to levels induced by bacterial cells, MV-stimulated macrophages and dendritic cells displayed increased surface expression of MHC-II and CD86 and enhanced production of the proinflammatory mediators NO, TNF-, and IL-12. MV-mediated dendritic cell stimulation occurred by TLR4-dependent and -independent signals, indicating the stimulatory properties of Salmonella MVs, which contain LPS, do not strictly rely on signaling through TLR4. In addition to their strong proinflammatory properties, MVs contained Ags recognized by Salmonella-specific B cells and CD4⁺ T cells; MV-vaccinated mice generated Salmonella-specific Ig and CD4⁺ T cell responses in vivo and were significantly protected from infectious challenge with live Salmonella. Our findings demonstrate that MVs possess important inflammatory properties as well as B and T cell Ags known to influence the development of Salmonella-specific immunity to infection in vivo. Our findings also reveal MVs are a functional nonviable complex vaccine for Salmonella by their ability to prime protective B and T cell responses in vivo.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grant AI47242 (to B.T.C.) and National Institutes of Health Bacterial Pathogenesis Training Grant AI055396 (to B.L.D.).

² Address correspondence and reprint requests to Dr. Brad T. Cookson, Department of Laboratory Medicine and Microbiology, University of Washington Medical Center, Box 357110, 1959 NE Pacific Street, Seattle, WA 98195. E-mail address: cookson{at}u.washington.edu

³ Abbreviations used in this paper: MV, membrane vesicle; DC, dendritic cell; HKST, heat-inactivated Salmonella Ag; ICS, intracellular cytokine staining; M, macrophage; NTS, nontyphoidal salmonellosis; OmpA, outer membrane protein A; PAMP, pathogen-associated molecular pattern.