title: Loss of CD40 endogenous S-nitrosylation during inflammatory response in endotoxemic mice and patients with sepsis.
abstract: Signal transduction through the surface molecule CD40 is critical for cellular activation in immunoinflammatory states such as sepsis. The mechanisms regulating this pathway are not completely understood. Because CD40 displays potentially regulatory cysteine residues and CD40 is probably exposed to NO in the inflammatory milieu, we hypothesized that S-nitrosylation, the interaction of NO with cysteines residues, acts as a post-translational modification on CD40, coregulating the signaling activity and, therefore, the level of cellular activation. As assessed by the biotin switch and the reduction/chemiluminescence S-nitrosylation detection techniques, CD40 was found to be S-nitrosylated endogenously and upon exposure to NO donors in both human and murine macrophages. S-nitrosylation of CD40 was associated with milder activation by its ligand (CD40L), leading to reduced in vitro cytokine (IL-1beta, IL-12, and TNF-alpha) production, which was reversed in the presence of inhibitors of NO synthesis. S-nitrosylated CD40 was found in resting RAW 246.7 macrophages and BALB/c mice peritoneal macrophages, turning into the denitrosylated state upon in vitro or systemic exposure, respectively, to LPS. Moreover, monocytes from patients with sepsis displayed denitrosylated CD40 in contrast to the CD40 S-nitrosylation measured in healthy individuals. Finally, in an attempt to explain how S-nitrosylation regulates CD40 activation, we demonstrate that NO affects the redistribution of CD40 on the cell surface, which is a requirement for optimal signal transduction. Our results support a novel post-translational regulatory mechanism in which the CD40 signal may be, at least in part, dependent on cellular activation-induced receptor denitrosylation.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. lcgodoy@mit.edu
authors.forename: Luiz C
authors.initials: LC
authors.lastname: Godoy
authors.forename: Ana I
authors.initials: AI
authors.lastname: Moretti
authors.forename: Marcia C
authors.initials: MC
authors.lastname: Jurado
authors.forename: Daniella
authors.initials: D
authors.lastname: Oxer
authors.forename: Mariano
authors.initials: M
authors.lastname: Janiszewski
authors.forename: Karina
authors.initials: K
authors.lastname: Ckless
authors.forename: Irineu T
authors.initials: IT
authors.lastname: Velasco
authors.forename: Francisco R M
authors.initials: FR
authors.lastname: Laurindo
authors.forename: Heraldo P
authors.initials: HP
authors.lastname: Souza
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D019013:CD40 Antigens / Q000378:metabolism*; D023201:CD40 Ligand / Q000378:metabolism; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis; D015536:Down-Regulation; D019446:Endotoxemia / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007249:Inflammation / Q000503:physiopathology*; D008264:Macrophages / Q000187:drug effects / Q000276:immunology; D051379:Mice; D008875:Middle Aged; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D018805:Sepsis / Q000276:immunology* / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology*
chemical_list: D019013:CD40 Antigens; D016207:Cytokines; D020030:Nitric Oxide Donors; D023201:CD40 Ligand; D009569:Nitric Oxide