title: Developmental onset of bilirubin-induced neurotoxicity involves Toll-like receptor 2-dependent signaling in humanized UDP-glucuronosyltransferase1 mice.
abstract: Biological and signaling events that connect developmentally induced hyperbilirubinemia to bilirubin-induced neurological dysfunction (BIND) and CNS toxicity in humans are poorly understood. In mammals, UDP-glucuronosyltransferase 1A1 (UGT1A1) is the sole enzyme responsible for bilirubin glucuronidation, a rate-limiting step necessary for bilirubin metabolism and clearance. Humanized mice that express the entire UGT1 locus (hUGT1) and the UGT1A1 gene, develop neonatal hyperbilirubinemia, with 8-10% of hUGT1 mice succumbing to CNS damage, a phenotype that is presented by uncontrollable seizures. We demonstrate that neuroinflammation and reactive gliosis are prominent features of bilirubin brain toxicity, and a disturbed redox status resulting from activation of NADPH oxidase is an important contributing mechanism found in BIND. Using knock-out mice and primary brain cells, we connect a key pattern recognition receptor, Toll-like receptor 2 (TLR2), to hyperbilirubinemia-induced signaling. We illustrate a requirement for TLR2 signaling in regulating gliosis, proinflammatory mediators, and oxidative stress when neonatal mice encounter severe hyperbilirubinemia. TLR2-mediated gliosis strongly correlates with pronounced neuroinflammation in the CNS with up-regulation of TNFα, IL-1β, and IL-6, creating a pro-inflammatory CNS environment. Gene expression and immunohistochemistry staining show that hUGT1/Tlr2(-/-) mice fail to activate glial cells, proinflammatory cytokines, and stress response genes. In addition, bilirubin-induced apoptosis was significantly enhanced by blocking TLR2 signaling indicating its anti-apoptotic property. Consequently, a higher neonatal death rate (57.1%) in hUGT1/Tlr2(-/-) mice was observed when compared with hUGT1 mice (8.7%). These results suggest that TLR2 signaling and microglia neuroinflammation are linked to a repair and/or protection mode against BIND.
authors.affiliation: From the Departments of Chemistry & Biochemistry and Pharmacology, Laboratory of Environmental Toxicology, University of California at San Diego, La Jolla, California 92093.
authors.forename: Mei-Fei
authors.initials: MF
authors.lastname: Yueh
authors.forename: Shujuan
authors.initials: S
authors.lastname: Chen
authors.forename: Nghia
authors.initials: N
authors.lastname: Nguyen
authors.forename: Robert H
authors.initials: RH
authors.lastname: Tukey
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001663:Bilirubin / Q000009:adverse effects* / Q000097:blood; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000187:drug effects; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism*; D006801:Humans; D006932:Hyperbilirubinemia / Q000097:blood / Q000150:complications / Q000235:genetics / Q000473:pathology; D018836:Inflammation Mediators / Q000378:metabolism; D007647:Kernicterus / Q000097:blood / Q000150:complications / Q000235:genetics / Q000473:pathology; D051379:Mice; D008822:Mice, Transgenic; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D020258:Neurotoxicity Syndromes / Q000097:blood / Q000150:complications / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013312:Stress, Physiological / Q000187:drug effects / Q000235:genetics; D051195:Toll-Like Receptor 2 / Q000172:deficiency / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000099148:UGT1A1 Enzyme
chemical_list: D001663:Bilirubin; D002135:Calcium-Binding Proteins; D051546:Cyclooxygenase 2; D005904:Glial Fibrillary Acidic Protein; D014453:Glucuronosyltransferase; D018836:Inflammation Mediators; D008840:Microfilament Proteins; D019255:NADPH Oxidases; D016328:NF-kappa B; D051195:Toll-Like Receptor 2; D014409:Tumor Necrosis Factor-alpha; D000099148:UGT1A1 Enzyme; C491049:Aif1 protein, mouse; C077623:Ptgs2 protein, mouse
keywords: Brain; Glia; Neuroinflammation; Oxidative Stress; Toll-like Receptors (TLR); UDP-glucuronosyltransferase