Effet des agonistes des TRL sur la production des FRO par la NADPH oxydase des polynucléaires neutrophiles humains

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 to 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 Ser345, Ser328 and Ser315. Phosphorylations of Ser328 and Ser315 were significantly increased in CL097-primed and fMLF-stimulated neutrophils. Phosphorylation of Ser345, Ser328 and Ser315 was decreased by inhibitors of p38MAPK and the ERK1/2-pathway. Phosphorylation of Ser328 was decreased by a PKC inhibitor. Genistein, a braod range protein tyrosine kinase inhibitor, inhibited the phosphorylation of these serines. Our results also show that CL097 induced proline isomerase (Pin1) activation and that juglone, a Pin1 inhibitor, inhibited CL097-mediated priming of fMLF-induced p47phox phosphorylation and superoxide production. These results show that activation of TLR7/8 in human neutrophils induces hyper-activation of the NADPH oxidase by stimulating the phosphorylation of p47phox on selective sites, and suggest that p38MAPK, ERK1/2, PKC and Pin1 control this process.Zymosan a cell-wall preparation from saccharomyces cerevisiae is largely used to activate neutrophils in its opsonized form. In this study, we show that non-opsonized zymosan induced ROS production by human neutrophils. Interestingly, zymosan induced the phosphorylation of the NADPH oxidase cytosolic component p47phox on Ser345, Ser328 and Ser315; and activation of the GTPase Rac2. Phosphorylation of p47phox as well as Rac2 activation were inhibited by genistein a broad range protein tyrosine kinase inhibitor. Wortmannin a PI3Kinase inhibitor, inhibited phosphorylation of p47phox on Ser328 and Ser315 and Rac2 activation. SB203580 and UO126, inhibitors of p38MAPK and ERK1/2-pathway respectively, inhibited phosphorylation of p47phox on Ser345. GF109203X a PKC inhibitor inhibited phosphorylation on Ser328 and Ser315. Zymosan-induced ROS production was inhibited by genistein, wortmannin, SB203580, UO126 and GF109203X. These results show that zymosan induced ROS production by NADPH oxidase in human neutrophils via the phosphorylation of p47phox and Rac2 activation. Our results also suggest that a protein tyrosine kinase and PI3Kinase control p47phox phosphorylation and Rac2 activation while p38MAPK, ERK1/2 and PKC are involved in zymosan-induced p47phox phosphorylation.
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Karama Makni Maalej. Effet des agonistes des TRL sur la production des FRO par la NADPH oxydase des polynucléaires neutrophiles humains. Immunologie. Université Paris Sud - Paris XI, 2012. Français. ⟨NNT : 2012PA11T037⟩. ⟨tel-01239582⟩

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