Bilirubin inhibits the up-regulation of inducible nitric oxide synthase by scavenging reactive oxygen species generated by the toll-like receptor 4-dependent activation of NADPH oxidase

It has been previously shown that bilirubin prevents the up-regulation of inducible nitric oxide synthase (iNOS) in response to LPS. The present study examines whether this effect is exerted through modulation of Toll-Like Receptor-4 (TLR4) signaling. LPS-stimulated iNOS and NADPH oxidase (Nox) activity in RAW 264.7 murine macrophages was assessed by measuring cellular nitrate and superoxide (O-2(-)) production, respectively. The generation of both nitrate and O-2(-) in response to LPS was suppressed by TLR4 inhibitors, indicating that activation of iNOS and Nox is TLR4-dependent. While treatment with superoxide dismutase (SOD) and bilirubin effectively abolished LPS-mediated O-2(-) production, hydrogen peroxide and nitrate release were inhibited by bilirubin and PEG-catalase, but not SOD, supporting that iNOS activation is primarily dependent upon intracellular H2O2. LPS treatment increased nuclear translocation of the redox-sensitive transcription factor Hypoxia Inducible Factor-1 alpha (HIF-1 alpha), an effect that was abolished by bilirubin. Cells transfected with murine iNOS reporter constructs in which the HIF-1 alpha-specific hypoxia response element was disrupted exhibited a blunted response to LPS, supporting that HIF-1 alpha mediates Nox-dependent iNOS expression. Bilirubin, but not SOD, blocked the cellular production of interferon-beta, while interleukin-6 production remained unaffected. These data support that bilirubin inhibits the TLR4-mediated up-regulation of iNOS by preventing activation of HIF-1 alpha through scavenging of Nox-derived reactive oxygen species. Bilirubin also suppresses interferon-beta release via a ROS-independent mechanism. These findings characterize potential mechanisms for the anti-inflammatory effects of bilirubin. (C) 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).