title: The TLR7/8 agonist CL097 primes N-formyl-methionyl-leucyl-phenylalanine-stimulated NADPH oxidase activation in human neutrophils: critical role of p47phox phosphorylation and the proline isomerase Pin1.
abstract: Superoxide anion production by the neutrophil NADPH oxidase plays a key role in host defense; however, excessive superoxide production is believed to participate to inflammatory reactions. Neutrophils express several TLR that recognize a variety of microbial motifs or agonists. The interaction between TLR and their agonists is believed to help neutrophils to recognize and eliminate the pathogen. However, the effects of some TLR agonists on the NADPH oxidase activation and the mechanisms controlling these effects have not been elucidated. In this study, we show that the TLR7/8 agonist CL097 by itself did not induce NADPH oxidase activation in human neutrophils, but induced a dramatic increase of fMLF-stimulated activation. Interestingly, CL097 induced cytochrome b558 translocation to the plasma membrane and the phosphorylation of the NADPH oxidase cytosolic component p47phox on Ser(345), Ser(328), and Ser(315). Phosphorylation of Ser(328) and Ser(315) was significantly increased in CL097-primed and fMLF-stimulated neutrophils. Phosphorylation of Ser(345), Ser(328), and Ser(315) was decreased by inhibitors of p38 MAPK and the ERK1/2 pathway. Phosphorylation of Ser(328) was decreased by a protein kinase C inhibitor. Genistein, a broad-range protein tyrosine kinase inhibitor, inhibited the phosphorylation of these serines. Our results also show that CL097 induced proline isomerase 1 (Pin1) activation and that juglone, a Pin1 inhibitor, inhibited CL097-mediated priming of fMLF-induced p47phox phosphorylation and superoxide production. These results show that the TLR7/8 agonist CL097 induces hyperactivation of the NADPH oxidase by stimulating the phosphorylation of p47phox on selective sites in human neutrophils and suggest that p38 MAPK, ERK1/2, protein kinase C, and Pin1 control this process.
authors.affiliation: INSERM, U773, Centre de Recherche Biomédicale Bichat Beaujon, Paris F-75018, France.
authors.forename: Karama
authors.initials: K
authors.lastname: Makni-Maalej
authors.forename: Tarek
authors.initials: T
authors.lastname: Boussetta
authors.forename: Margarita
authors.initials: M
authors.lastname: Hurtado-Nedelec
authors.forename: Sahra Amel
authors.initials: SA
authors.lastname: Belambri
authors.forename: Marie-Anne
authors.initials: MA
authors.lastname: Gougerot-Pocidalo
authors.forename: Jamel
authors.initials: J
authors.lastname: El-Benna
mesh_terms: D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects / Q000276:immunology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology* / Q000627:therapeutic use; D007249:Inflammation / Q000201:enzymology / Q000276:immunology / Q000517:prevention & control; D058990:Molecular Targeted Therapy / Q000379:methods; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000378:metabolism* / Q000502:physiology; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D009504:Neutrophils / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects / Q000276:immunology; D011804:Quinolines / Q000494:pharmacology* / Q000627:therapeutic use; D017382:Reactive Oxygen Species / Q000037:antagonists & inhibitors / Q000378:metabolism; D051199:Toll-Like Receptor 7 / Q000819:agonists*; D051200:Toll-Like Receptor 8 / Q000819:agonists*
chemical_list: C547051:CL097 compound; D007093:Imidazoles; D000072340:NIMA-Interacting Peptidylprolyl Isomerase; D011804:Quinolines; D017382:Reactive Oxygen Species; C495348:TLR7 protein, human; C495349:TLR8 protein, human; D051199:Toll-Like Receptor 7; D051200:Toll-Like Receptor 8; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; C000606846:PIN1 protein, human; D019696:Peptidylprolyl Isomerase