title: Differential activation of human TLR4 by Escherichia coli and Shigella flexneri 2a lipopolysaccharide: combined effects of lipid A acylation state and TLR4 polymorphisms on signaling.
abstract: 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-kappaB-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-kappaB 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-alpha, IL-6, IL-1beta, and IL-10 production. Cytokine levels were significantly lower (approximately 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.
authors.affiliation: Department of Microbiology and Immunology, Center for Vaccine Development, University of Maryland, Baltimore 21201, USA.
authors.forename: Prasad
authors.initials: P
authors.lastname: Rallabhandi
authors.forename: Agnes
authors.initials: A
authors.lastname: Awomoyi
authors.forename: Karen E
authors.initials: KE
authors.lastname: Thomas
authors.forename: Armelle
authors.initials: A
authors.lastname: Phalipon
authors.forename: Yukari
authors.initials: Y
authors.lastname: Fujimoto
authors.forename: Koichi
authors.initials: K
authors.lastname: Fukase
authors.forename: Shoichi
authors.initials: S
authors.lastname: Kusumoto
authors.forename: Nilofer
authors.initials: N
authors.lastname: Qureshi
authors.forename: Marcelo B
authors.initials: MB
authors.lastname: Sztein
authors.forename: Stefanie N
authors.initials: SN
authors.lastname: Vogel
mesh_terms: D000215:Acylation; D019943:Amino Acid Substitution; D000818:Animals; D001428:Bacterial Vaccines / Q000276:immunology; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D004926:Escherichia coli / Q000276:immunology*; D017930:Genes, Reporter; D006801:Humans; D008050:Lipid A / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000276:immunology* / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D012763:Shigella flexneri / Q000276:immunology*; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000819:agonists* / Q000235:genetics*; D014162:Transfection
chemical_list: D001428:Bacterial Vaccines; D016207:Cytokines; D008050:Lipid A; D008070:Lipopolysaccharides; D016328:NF-kappa B; D051197:Toll-Like Receptor 4; D008156:Luciferases