Division of Cardiology, Department of Medicine, The Affiliated People's Hospital of Jiangsu University, Zhenjiang, China.
Critical Illness Research, Lawson Health Research Institute, London, ON, Canada.
Front Immunol. 2020 Sep 25;11:558712. doi: 10.3389/fimmu.2020.558712. eCollection 2020.
Sepsis/endotoxemia activates the NLRP3 inflammasome of macrophages leading to the maturation and release of IL-1β, 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 β. 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). Isolated murine fibroblasts were primed with LPS (1 μg/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-1β protein (Western) were used as readouts for inflammasome priming, while activation of caspase 1 (p20) (Western) and secretion of IL-1β (ELISA) were indicative of inflammasome activation. Inhibition of PKR (PKR inhibitor or siRNA) prior to priming with LPS prevented the LPS-induced increase in NLRP3 and pro-IL-1β expression. Further, inhibition of PKR after priming, but before activation, did not affect NLRP3 or pro-IL-1β protein levels, but markedly reduced the activation of caspase 1 and secretion of mature IL-1β. 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). Our results indicate that peroxynitrite-/PKR pathway modulates priming and activation of NLRP3 inflammasome in LPS/ATP challenged cardiac fibroblasts.
脓毒症/内毒素血症激活巨噬细胞中的 NLRP3 炎性体，导致白细胞介素-1β（IL-1β）的成熟和释放，这是炎症反应的重要介质。活性氧被认为参与了 NLRP3 炎性体的激活。此外，我们的初步研究表明，LPS 可使心脏成纤维细胞中的蛋白激酶 R（PKR）磷酸化，并增加前体 IL-1β 的 mRNA 表达。因此，本研究的主要目的是探讨 PKR 和氧化剂过氧亚硝酸盐在 NLRP3 炎性体（预激活和激活）的双重功能中的作用。将分离的鼠成纤维细胞用 LPS（1μg/ml）预激活 6 小时，然后用 ATP（3mM）刺激 30 分钟以诱导炎性体的最佳功能。NLRP3 和前体 IL-1β 蛋白（Western）水平的增加被用作炎性体预激活的读数，而 caspase 1（p20）的激活（Western）和 IL-1β 的分泌（ELISA）则表示炎性体的激活。在 LPS 预激活之前抑制 PKR（PKR 抑制剂或 siRNA）可阻止 LPS 诱导的 NLRP3 和前体 IL-1β 表达的增加。此外，在预激活后但在激活之前抑制 PKR 不会影响 NLRP3 或前体 IL-1β 蛋白水平，但明显减少 caspase 1 的激活和成熟的 IL-1β 的分泌。同样，过氧亚硝酸盐分解催化剂（Fe-TPPS）可阻止 NLRP3 炎性体的预激活和激活。最后，成纤维细胞的 Fe-TPPS 预处理可防止 LPS 诱导的 PKR 磷酸化（T451）。我们的结果表明，过氧亚硝酸盐/PKR 途径调节 LPS/ATP 刺激的心脏成纤维细胞中 NLRP3 炎性体的预激活和激活。
