COVID-19 airway inflammation is due to Spike inhibition of CFTR signaling

摘要 美国已有超过860,000人死于新冠肺炎，550多万人死于 发生在世界各地。因此，有效的抗病毒药物的开发可以阻断传染性和呼吸道 炎症仍然是全球卫生优先事项。新冠肺炎的炎症是由于增加了 核因子κB与上皮钠通道信号转导。囊性纤维化是一种遗传性疾病，由 Cftr基因的失活突变，也具有相同的促炎因子κB和ENaC信号 呼吸道的表型。利用一个分化的人类上皮细胞“芯片上肺”平台，我们发现ACE2， SARS-CoV-2刺突蛋白的受体蛋白与CFTR共免疫沉淀。此外， SARS-COV-2刺突蛋白对分化上皮细胞周期的抑制作用呈剂量依赖性 依赖AMP的CFTR氯通道活性和CFTR蛋白表达。CFTR的尖峰相关损耗 也激活TRANDD依赖的NFκB信号和蛋白水解性激活ENaC。我们还发现， 尖峰相关的cftr丢失可能是由于内体循环不能将心尖cftr返回到 质膜。最后，我们发现纳摩尔浓度的心脏糖苷药物，如 哇巴因、洋地黄毒和地高辛竞争性抑制Spike：ACE2结合，挽救Spike依赖 减少商品期货交易委员会的活动。因此，我们假设SARS-CoV-2刺突蛋白与 肺内血管紧张素转换酶2导致cftr信号的丢失以及促炎因子κB和eNaC的激活 发信号。为了进一步检验这一假设，我们提出了以下具体目标：SA#1：定义 SARS-CoV-2刺突蛋白降低CFTR通道活性及其蛋白机制的研究 级别。我们将确定ACE2与CFTR相互作用的机制。我们将确定 Spike蛋白与ACE2相互作用导致cftr缺失的机制SA#2：定义 SARS冠状病毒S蛋白激活ENaC和NFκB信号的机制我们会 确定SPEKE诱导的CFTR蛋白丢失导致Tradd激活的机制 依赖的NFκB ENaC信号转导。SA#3：确定心脏糖苷对细胞的保护机制 以及新冠肺炎病的动物模型。我们将测试心苷类药物是否能阻断本土SARS-- 新冠肺炎上皮细胞冠状病毒2型感染及救治仓鼠模型。。 新颖性和意义：据我们所知，这是新冠肺炎首次出现呼吸道炎症 可追溯到对CFTR信号的抑制。一如既往，新冠肺炎患者也是CF携带者，只有一人 野生型cftr基因和仅50%的cftr功能，最近被报道遭受更严重的新冠肺炎 与正常新冠肺炎受试者相比，患者的症状和死亡时间更早。

Abstract Over 860,000 COVID-19 deaths have occurred in the U.S., and more than 5.5 million deaths have occurred world-wide. Consequently, development of effective antiviral drugs that block infectivity and airway inflammation continue to be a global health priority. Inflammation in the COVID-19 airway is due to increased NFκB and Epithelial Sodium channel (ENaC) signaling. Cystic fibrosis (CF), a genetic disease caused by inactivating mutations in the CFTR gene, also has the same proinflammatory NFκB and ENaC signaling phenotype in the airway. Using a differentiated human epithelial "lung-on-a-chip" platform, we find that ACE2, the receptor protein for the SARS-CoV-2 Spike protein, co-immunoprecipitates with CFTR. Furthermore, exposure of differentiated epithelia to the SARS-COV-2 Spike protein dose-dependently suppresses cyclic AMP-dependent CFTR chloride channel activity and CFTR protein expression. Spike-dependent loss of CFTR also activates TRADD-dependent NFκB signaling and proteolytically activates ENaC. We have also found that Spike-dependent loss of CFTR may be due to failure of endosomal recycling to return apical CFTR to the plasma membrane. Finally, we find that nanoMolar concentrations of cardiac glycoside drugs such as ouabain, digitoxin and digoxin, which competitively inhibit Spike:ACE2 binding, rescue Spike-dependent reduction in CFTR activities. We have therefore hypothesized that binding of SARS-CoV-2 Spike protein to ACE2 in the lung causes loss of CFTR signaling and activation of proinflammatory NFκB and ENaC signaling. To further test this hypothesis we propose the following Specific Aims: SA #1: To define the mechanism by which SARS-CoV-2 Spike protein reduces CFTR channel activity and CFTR protein levels. We will determine the mechanism by which ACE2 interacts with CFTR. We will determine the mechanism by which Spike protein interaction with ACE2 leads to loss of CFTR. SA#2: To define the mechanism by which SARS-CoV-2 Spike protein drives activation of ENaC and NFκB signaling. We will determine the mechanism by which Spike-induced loss of CFTR protein results in activation of TRADD- dependent NFκB ENaC signaling. SA#3: To identify protective mechanisms of cardiac glycosides on cell and animal models of COVID-19 disease. We will test whether cardiac glycoside drugs block native SARS- CoV-2 infection of epithelia and rescue hamster models of COVID-19. . Novelty and Significance: To our knowledge this is the first time COVID-19 airway inflammation has been traced to inhibition of CFTR signaling. Consistently, COVID-19 patients who are also CF carriers, with only one wildtype CFTR gene and only 50% of CFTR function, were recently reported to suffer more severe COVID-19 symptoms and earlier death than normal subjects with COVID-19.