背景:严重急性呼吸道综合征冠状病毒2(SARS-CoV-2)的刺突蛋白对病毒进入至关重要,并可引起心脏损伤。Toll样受体4(TLR4)和NOD-,LPR-,和含pyrin结构域的3(NLRP3)炎性体是与心脏纤维化有关的关键免疫系统成分。刺突蛋白通过TLR4或血管紧张素转换酶2(ACE2)受体激活NLRP3炎性体,损害各种器官。然而,刺突蛋白在人类心脏纤维化中的作用,以及它与NLRP3炎性体和TLR4的相互作用,仍然知之甚少。
方法:我们利用划痕试验,西方印迹,和免疫荧光来评估迁移,纤维化信号,线粒体钙水平,活性氧(ROS)的产生,和培养的人心脏成纤维细胞(CFs)的细胞形态用刺突(S1)蛋白处理24小时,有或没有抗ACE2中和抗体,TLR4阻断剂,或NLRP3抑制剂。
结果:S1蛋白增强CFs的迁移和胶原1、α-平滑肌肌动蛋白的表达,转化生长因子β1(TGF-β1),磷酸化SMAD2/3,白细胞介素1β(IL-1β),和活化B细胞的核因子κ-轻链增强子(NF-κB)。S1蛋白增加了ROS的产生,但不影响线粒体钙含量和细胞形态。用抗ACE2中和抗体处理减弱了S1蛋白对胶原蛋白1和TGF-β1表达的影响。此外,NLRP3(MCC950)和NF-kB抑制剂,但TLR4抑制剂TAK-242不能阻止S1蛋白增强的CFs迁移和胶原蛋白1,TGF-β1和IL-1β的过表达。
结论:S1蛋白通过NF-κB信号启动NLRP3炎性体以ACE2依赖性方式激活人类CFs。
BACKGROUND: The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to viral entry and can cause cardiac injuries. Toll-like receptor 4 (TLR4) and NOD-, LPR-, and pyrin-domain-containing 3 (NLRP3) inflammasome are critical immune system components implicated in cardiac fibrosis. The spike protein activates NLRP3 inflammasome through TLR4 or angiotensin-converting enzyme 2 (ACE2) receptors, damaging various organs. However, the role of spike protein in cardiac fibrosis in humans, as well as its interactions with NLRP3 inflammasomes and TLR4, remain poorly understood.
METHODS: We utilized scratch assays, Western blotting, and immunofluorescence to evaluate the migration, fibrosis signaling, mitochondrial calcium levels, reactive oxygen species (ROS) production, and cell morphology of cultured human cardiac fibroblasts (CFs) treated with spike (S1) protein for 24 h with or without an anti-ACE2 neutralizing antibody, a TLR4 blocker, or an NLRP3 inhibitor.
RESULTS: S1 protein enhanced CFs migration and the expressions of collagen 1, α-smooth muscle actin, transforming growth factor β1 (TGF-β1), phosphorylated SMAD2/3, interleukin 1β (IL-1β), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). S1 protein increased ROS production but did not affect mitochondrial calcium content and cell morphology. Treatment with an anti-ACE2 neutralizing antibody attenuated the effects of S1 protein on collagen 1 and TGF-β1 expressions. Moreover, NLRP3 (MCC950) and NF-kB inhibitors, but not the TLR4 inhibitor TAK-242, prevented the S1 protein-enhanced CFs migration and overexpression of collagen 1, TGF-β1, and IL-1β.
CONCLUSIONS: S1 protein activates human CFs by priming NLRP3 inflammasomes through NF-κB signaling in an ACE2-dependent manner.