title: Endothelial cell tolerance to lipopolysaccharide challenge is induced by monophosphoryl lipid A.
abstract: Prior exposure to lipopolysaccharide (LPS) produces a reduced or "tolerant" inflammatory response to subsequent challenges with LPS, however the potent pro-inflammatory effects of LPS limit its clinical benefit. The adjuvant monophosphoryl lipid A (MPLA) is a weak toll-like receptor 4 (TLR4) agonist that induces negligible inflammation but retains potent immunomodulatory properties. We postulated that pre-treatment with MPLA would inhibit the inflammatory response of endothelial cells to secondary LPS challenge. Human umbilical vein endothelial cells (HUVECs), were exposed to MPLA (10 μg/ml), LPS (100 ng/ml) or vehicle control. HUVECs were then washed and maintained in culture for 24 h before being challenged with LPS (100 ng/ml). Supernatants were collected and examined for cytokine production in the presence or absence of siRNA inhibitors of critical TLR4 signalling proteins. Pre-treatment with MPLA attenuated interleukin (IL)-6 production to secondary LPS challenge to a similar degree as LPS. The application of myeloid differentiation primary response gene 88 (MyD88) siRNA dramatically reduced MPLA-induced tolerance while TIR-domain-containing adapter-inducing interferon-β (TRIF) siRNA had no effect. The tolerant phenotype in endothelial cells was associated with reduced IκB kinase (IKK), p38 and c-Jun N-terminal kinase (JNK) phosphorylation and enhanced IL-1 receptor associated kinase-M (IRAK-M) expression for LPS-primed HUVECs, but less so in MPLA primed cells. Instead, MPLA-primed HUVECs demonstrated enhanced p-extracellular-signal-regulated kinase (ERK) phosphorylation. In contrast with leucocytes in which tolerance is largely TRIF-dependent, MyD88 signalling mediated endotoxin tolerance in endothelial cells. Most importantly, MPLA, a vaccine adjuvant with a wide therapeutic window, induced tolerance to LPS in endothelial cells.
authors.affiliation: Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, U.S.A. ryan.stark@vanderbilt.edu.
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Stark
authors.affiliation: Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, U.S.A.
authors.forename: Hyehun
authors.initials: H
authors.lastname: Choi
authors.affiliation: Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, U.S.A.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Koch
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, U.S.A.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Fensterheim
authors.affiliation: Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN 37232, U.S.A.
authors.forename: Fred S
authors.initials: FS
authors.lastname: Lamb
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, U.S.A. Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, TN 37232, U.S.A.
authors.forename: Edward R
authors.initials: ER
authors.lastname: Sherwood
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007108:Immune Tolerance / Q000187:drug effects; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism; D008050:Lipid A / Q000031:analogs & derivatives* / Q000494:pharmacology; D008070:Lipopolysaccharides; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D000276:Adjuvants, Immunologic; D008050:Lipid A; D008070:Lipopolysaccharides; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; C470409:TICAM1 protein, human; C507771:IRAK3 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; C048436:monophosphoryl lipid A
keywords: endothelial cells; endotoxin; monophosphoryl lipid A; tolerance; toll-like receptor 4