title: Hemorrhagic shock induces NAD(P)H oxidase activation in neutrophils: role of HMGB1-TLR4 signaling.
abstract: Hemorrhagic shock/resuscitation (HS/R)-induced generation of reactive oxygen species (ROS) plays an important role in posthemorrhage inflammation and tissue injury. We have recently reported that HS/R-activated neutrophils (PMN), through release of ROS, serve an important signaling function in mediating alveolar macrophage priming and lung inflammation. PMN NAD(P)H oxidase has been thought to be an important source of ROS following HS/R. TLR4 sits at the interface of microbial and sterile inflammation by mediating responses to both bacterial endotoxin and multiple endogenous ligands, including high-mobility group box 1 (HMGB1). Recent studies have implicated HMGB1 as an early mediator of inflammation after HS/R and organ ischemia/reperfusion. In the present study, we tested the hypothesis that HS/R activates NAD(P)H oxidase in PMN through HMGB1/TLR4 signaling. We demonstrated that HS/R induced PMN NAD(P)H oxidase activation, in the form of phosphorylation of p47phox subunit of NAD(P)H oxidase, in wild-type mice; this induction was significantly diminished in TLR4-mutant C3H/HeJ mice. HMGB1 levels in lungs, liver, and serum were increased as early as 2 h after HS/R. Neutralizing Ab to HMGB1 prevented HS/R-induced phosphorylation of p47phox in PMN. In addition, in vitro stimulation of PMN with recombinant HMGB1 caused TLR4-dependent activation of NAD(P)H oxidase as well as increased ROS production through both MyD88-IRAK4-p38 MAPK and MyD88-IRAK4-Akt signaling pathways. Thus, PMN NAD(P)H oxidase activation, induced by HS/R and as mediated by HMGB1/TLR4 signaling, is an important mechanism responsible for PMN-mediated inflammation and organ injury after hemorrhage.
authors.affiliation: Department of Surgery, School of Medicine, University of Pittsburgh, and Division of Pediatric Surgery, Children's Hospital of Pittsburgh, PA 15213, USA. jif7@pitt.edu
authors.forename: Jie
authors.initials: J
authors.lastname: Fan
authors.forename: Yuehua
authors.initials: Y
authors.lastname: Li
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Levy
authors.forename: Janet J
authors.initials: JJ
authors.lastname: Fan
authors.forename: David J
authors.initials: DJ
authors.lastname: Hackam
authors.forename: Yoram
authors.initials: Y
authors.lastname: Vodovotz
authors.forename: Huan
authors.initials: H
authors.lastname: Yang
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Tracey
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Billiar
authors.forename: Mark A
authors.initials: MA
authors.lastname: Wilson
mesh_terms: D000818:Animals; D004789:Enzyme Activation / Q000276:immunology; D024243:HMGB1 Protein / Q000502:physiology*; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D019255:NADPH Oxidases / Q000096:biosynthesis / Q000378:metabolism*; D018375:Neutrophil Activation / Q000276:immunology*; D015427:Reperfusion Injury / Q000201:enzymology / Q000276:immunology / Q000473:pathology; D012151:Resuscitation; D012771:Shock, Hemorrhagic / Q000201:enzymology* / Q000276:immunology* / Q000473:pathology; D015398:Signal Transduction / Q000276:immunology*; D051197:Toll-Like Receptor 4 / Q000502:physiology*
chemical_list: D024243:HMGB1 Protein; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D019255:NADPH Oxidases