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* Department of Microbiology and Immunology and
Center for Vaccine Development, University of Maryland, Baltimore, MD 21201;
Institute Pasteur, Paris, France;
Osaka University, Osaka, Japan; and
¶ Department of Basic Medical Science, School of Medicine, University of Missouri, Kansas City, MO 64108
The lipid A of LPS activates TLR4 through an interaction with myeloid differentiation protein-2 (MD-2) and the degree of lipid A acylation affects TLR4 responsiveness. Two TLR4 single nucleotide polymorphisms (Asp299Gly and Thr399Ile) have been associated with LPS hyporesponsiveness. We hypothesized that the combination of hypoacylation and these single nucleotide polymorphisms would exhibit a compounded effect on TLR4 signaling. HEK293T transfectants expressing wild-type or polymorphic TLR4 were stimulated with Escherichia coli (predominantly hexaacylated lipid A) or Shigella flexneri 2a (a mixture of hexaacylated, pentaacylated, and predominantly tetraacylated lipid A) LPS, or hexaacylated vs pentaacylated synthetic lipid As. NF-
B-reporter activity was significantly lower in response to S. flexneri 2a than E. coli LPS and further decreased in polymorphic transfectants. Neither hexaacylated nor pentaacylated synthetic lipid A induced NF-B activity in wild-type transfectants under the identical transfection conditions used for LPS; however, increasing human MD-2 expression rescued responsiveness to hexaacylated lipid A only, while murine MD-2 was required to elicit a response to pentaacylated lipid A. Adherent PBMC of healthy volunteers were also compared for LPS-induced TNF-
, IL-6, IL-1β, and IL-10 production. Cytokine levels were significantly lower (
20–90%) in response to S. flexneri than to E. coli LPS/lipid A and PBMC from polymorphic individuals secreted decreased cytokine levels in response to both LPS types and failed to respond to pentaacylated lipid A. Thus, the combination of acylation state and host genetics may significantly impact vaccine immunogenicity and/or efficacy, whether LPS is an integral component of a whole organism vaccine or included as an adjuvant.
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, in part, with federal funds from National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. N01-AI-30028 (to M.B.S.) and Grants AI057927 (to M.B.S.), AI18797 (to S.N.V.), and GM50870 (to N.Q.).
2 P.R. and A.A. contributed equally to this study.
3 M.B.S. and S.N.V. contributed equally to the direction of this work.
4 Address correspondence and reprint requests to Dr. Stefanie N. Vogel, Department of Microbiology and Immunology, University of Maryland, 660 West Redwood Street, Room 324, Baltimore, MD 21201. E-mail address: svogel{at}som umaryland.edu
5 Abbreviations used in this paper: SNP, single nucleotide polymorphism; WT, wild type; hu, human; mu, murine; RLU, relative luciferase unit; HEK, human embryonic kidney; MS, mass spectroscopy; MD-2, myeloid differentiation protein-2.
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