Peroxynitrite/PKR Axis Modulates the NLRP3 Inflammasome of Cardiac Fibroblasts.

ABSTRACT: Sepsis/endotoxemia activates the NLRP3 inflammasome of macrophages leading to the maturation and release of IL-1?, an important mediator of the inflammatory response. Reactive oxygen species have been implicated in NLRP3 inflammasome activation. Further, our preliminary studies indicated that LPS challenge of cardiac fibroblasts could phosphorylate protein kinase R (PKR) on threonine 451 and increase message for pro-IL-1 ?. Thus, the major aim of the present study was to address the role of PKR and the oxidant, peroxynitrite, in the two-tiered function of the NLRP3 inflammasome (priming and activation). Materials and Methods: Isolated murine fibroblasts were primed with LPS (1 ?g/ml) for 6 h and subsequently activated by an ATP (3 mM) challenge for 30 min to induce optimum functioning of the inflammasome. Increased levels of NLRP3 and pro-IL-1? protein (Western) were used as readouts for inflammasome priming, while activation of caspase 1 (p20) (Western) and secretion of IL-1? (ELISA) were indicative of inflammasome activation. Results: Inhibition of PKR (PKR inhibitor or siRNA) prior to priming with LPS prevented the LPS-induced increase in NLRP3 and pro-IL-1? expression. Further, inhibition of PKR after priming, but before activation, did not affect NLRP3 or pro-IL-1? protein levels, but markedly reduced the activation of caspase 1 and secretion of mature IL-1?. In a similar fashion, a peroxynitrite decomposition catalyst (Fe-TPPS) prevented both the priming and activation of the NLRP3 inflammasome. Finally, pretreatment of the fibroblasts with Fe-TPPS prevented the LPS-induced PKR phosphorylation (T451). Conclusion: Our results indicate that peroxynitrite-/PKR pathway modulates priming and activation of NLRP3 inflammasome in LPS/ATP challenged cardiac fibroblasts.

SUBMITTER: Lan T 

PROVIDER: S-EPMC7545724 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature