title: Pleiotropic effects of 4-hydroxynonenal on oxidative burst and phagocytosis in neutrophils.
abstract: Metabolic control of cellular function is significant in the context of inflammation-induced metabolic dysregulation in immune cells. Generation of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide are one of the critical events that modulate the immune response in neutrophils. When activated, neutrophil NADPH oxidases consume large quantities of oxygen to rapidly generate ROS, a process that is referred to as the oxidative burst. These ROS are required for the efficient removal of phagocytized cellular debris and pathogens. In chronic inflammatory diseases, neutrophils are exposed to increased levels of oxidants and pro-inflammatory cytokines that can further prime oxidative burst responses and generate lipid oxidation products such as 4-hydroxynonenal (4-HNE). In this study we hypothesized that since 4-HNE can target glycolysis then this could modify the oxidative burst. To address this the oxidative burst was determined in freshly isolated healthy subject neutrophils using 13-phorbol myristate acetate (PMA) and the extracellular flux analyzer. Neutrophils pretreated with 4-HNE exhibited a significant decrease in the oxidative burst response and phagocytosis. Mass spectrometric analysis of alkyne-HNE treated neutrophils followed by click chemistry detected modification of a number of cytoskeletal, metabolic, redox and signaling proteins that are critical for the NADPH oxidase mediated oxidative burst. These modifications were confirmed using a candidate immunoblot approach for critical proteins of the active NADPH oxidase enzyme complex (Nox2 gp91phox subunit and Rac1 of the NADPH oxidase) and glyceraldehyde phosphate dehydrogenase, a critical enzyme in the metabolic regulation of oxidative burst. Taken together, these data suggest that 4-HNE-induces a pleiotropic mechanism to inhibit neutrophil function. These mechanisms may contribute to the immune dysregulation associated with chronic pathological conditions where 4-HNE is generated.
authors.affiliation: Mitochondrial Medicine Laboratory, University of Alabama at Birmingham, United States; Department of Pathology, University of Alabama at Birmingham, United States.
authors.forename: Balu K
authors.initials: BK
authors.lastname: Chacko
authors.affiliation: Department of Pathology, University of Alabama at Birmingham, United States.
authors.forename: Stephanie B
authors.initials: SB
authors.lastname: Wall
authors.affiliation: Mitochondrial Medicine Laboratory, University of Alabama at Birmingham, United States; Department of Pathology, University of Alabama at Birmingham, United States.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Kramer
authors.affiliation: Mitochondrial Medicine Laboratory, University of Alabama at Birmingham, United States; Department of Pathology, University of Alabama at Birmingham, United States.
authors.forename: Saranya
authors.initials: S
authors.lastname: Ravi
authors.affiliation: Department of Urology, University of Alabama at Birmingham, United States.
authors.forename: Tanecia
authors.initials: T
authors.lastname: Mitchell
authors.affiliation: Mitochondrial Medicine Laboratory, University of Alabama at Birmingham, United States; Department of Pathology, University of Alabama at Birmingham, United States.
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Johnson
authors.affiliation: Department of Pharmacology and Toxicology, The Targeted Metabolomics and Proteomics Laboratory, University of Alabama at Birmingham, United States.
authors.forename: Landon
authors.initials: L
authors.lastname: Wilson
authors.affiliation: Department of Pharmacology and Toxicology, The Targeted Metabolomics and Proteomics Laboratory, University of Alabama at Birmingham, United States.
authors.forename: Stephen
authors.initials: S
authors.lastname: Barnes
authors.affiliation: Department of Pathology, University of Alabama at Birmingham, United States.
authors.forename: Aimee
authors.initials: A
authors.lastname: Landar
authors.affiliation: Mitochondrial Medicine Laboratory, University of Alabama at Birmingham, United States; Department of Pathology, University of Alabama at Birmingham, United States. Electronic address: darley@uab.edu.
authors.forename: Victor M
authors.initials: VM
authors.lastname: Darley-Usmar
mesh_terms: D000328:Adult; D000447:Aldehydes / Q000494:pharmacology*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D006019:Glycolysis / Q000187:drug effects; D064368:Healthy Volunteers; D006801:Humans; D015227:Lipid Peroxidation / Q000187:drug effects; D008875:Middle Aged; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects* / Q000502:physiology*; D010587:Phagocytosis / Q000187:drug effects* / Q000276:immunology; D017382:Reactive Oxygen Species / Q000378:metabolism; D016897:Respiratory Burst / Q000187:drug effects* / Q000276:immunology
chemical_list: D000447:Aldehydes; D003598:Cytoskeletal Proteins; D017382:Reactive Oxygen Species; D019255:NADPH Oxidases; C027576:4-hydroxy-2-nonenal
keywords: Hydroxynonenal; Inflammation; Lipid peroxidation; Metabolism; NADPH Oxidase; Neutrophils; Oxidative burst; Oxidative stress