title: Nitric oxide production by endotoxin preparations in TLR4-deficient mice.
abstract: Sepsis and septic shock result from an exacerbated systemic inflammatory reaction to infection. Their incidence is rising, and they have recently become the main cause of death in intensive care units. Septic shock is defined as sepsis accompanied by life-threatening refractory hypotension, for which excessive nitric oxide (NO), produced by inducible NO synthase iNOS, is thought responsible. LPS, a vital outer membrane component of Gram-negative bacteria, mimics most of the septic effects and is widely used as a model for septic shock. TLR4 is the signal-transducing receptor for LPS, evidenced by the resistance of TLR4-deficient C3H/HeJ and C57BL/10ScNJ mice. As expected, we found that TLR4 deficiency precludes LPS-induced cytokine production, independent of the purity of the LPS preparation. However, various conventional LPS preparations induced NO in TLR4-deficient mice to the same level as in control animals, while ultrapure LPS did not, indicating the presence of NO-producing contaminant(s). Nevertheless, despite identical iNOS induction pattern and systemic NO levels, the contaminant does not cause hypotension, hypothermia, or any other sign of morbidity. Using mice deficient for TLR2, TRL3, TLR4, TRL2x4, TLR9, MyD88 or TRIF, we found that the contaminant signals via TLR2 and MyD88. In conclusion, conventional LPS preparations generally used in endotoxic shock research contain TLR2 agonists that induce iNOS and high levels of systemic NO as such, and synergize with LPS towards the production of pro-inflammatory cytokines, morbidity and mortality. Surprisingly, the excessive iNOS-derived systemic NO production induced by impure LPS in TLR4⁻/⁻ is not accompanied by hypotension or morbidity.
authors.affiliation: Department for Molecular Biomedical Research, VIB, B-9052 Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, B-9052 Ghent, Belgium. Electronic address: Anje.Cauwels@dmbr.UGent.be.
authors.forename: Anje
authors.initials: A
authors.lastname: Cauwels
authors.affiliation: Department for Molecular Biomedical Research, VIB, B-9052 Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, B-9052 Ghent, Belgium.
authors.forename: Jennyfer
authors.initials: J
authors.lastname: Bultinck
authors.affiliation: Department for Molecular Biomedical Research, VIB, B-9052 Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, B-9052 Ghent, Belgium.
authors.forename: Renske
authors.initials: R
authors.lastname: De Zwaef
authors.affiliation: Department for Molecular Biomedical Research, VIB, B-9052 Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, B-9052 Ghent, Belgium.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Vandendriessche
authors.affiliation: Department of Molecular and Cellular Interactions, VIB, B-1050 Brussels, Belgium; Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, B-1050 Brussels, Belgium.
authors.forename: Stefan
authors.initials: S
authors.lastname: Magez
authors.affiliation: Department for Molecular Biomedical Research, VIB, B-9052 Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, B-9052 Ghent, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Brouckaert
mesh_terms: D000818:Animals; D004731:Endotoxins / Q000378:metabolism*; D007022:Hypotension / Q000235:genetics; D007249:Inflammation; D008070:Lipopolysaccharides / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D018805:Sepsis / Q000378:metabolism*; D015398:Signal Transduction; D051195:Toll-Like Receptor 2 / Q000235:genetics; D051197:Toll-Like Receptor 4 / Q000235:genetics*
chemical_list: D004731:Endotoxins; D008070:Lipopolysaccharides; C493486:Tlr2 protein, mouse; C493487:Tlr4 protein, mouse; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse
keywords: Cardiovascular; Hypotension; LPS; NO; Shock