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

摘要 美国已经有超过86万人死于新冠肺炎，超过550万人死亡 发生在世界各地。因此，开发有效的抗病毒药物， 炎症仍然是全球卫生优先事项。COVID-19气道炎症是由于 NFκB和上皮钠通道（ENaC）信号传导。囊性纤维化（CF）是一种遗传性疾病， CFTR基因的失活突变，也具有相同的促炎性NFκB和ENaC信号传导 表型在气道中。使用分化的人上皮“芯片上的肺”平台，我们发现ACE 2， SARS-CoV-2刺突蛋白的受体蛋白与CFTR共免疫沉淀。此外，委员会认为， 将分化的上皮细胞暴露于SARS-COV-2刺突蛋白剂量依赖性地抑制了周期性细胞凋亡。 AMP依赖性CFTR氯离子通道活性和CFTR蛋白表达。CFTR的尖峰依赖性损失 还激活TRADD依赖性NFκB信号传导并蛋白水解激活ENaC。我们还发现 CFTR的尖峰依赖性损失可能是由于内体再循环失败，无法将顶端CFTR返回到 质膜最后，我们发现纳摩尔浓度的强心苷类药物， 哇巴因、洋地黄毒苷和地高辛，竞争性抑制刺突：ACE 2结合，拯救刺突依赖性 减少CFTR活动。因此，我们假设SARS-CoV-2刺突蛋白与 肺中的ACE 2导致CFTR信号传导丢失以及促炎性NFκB和ENaC的激活 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： SARS-CoV-2刺突蛋白降低CFTR通道活性和CFTR蛋白的机制 程度.我们将确定ACE 2与CFTR相互作用的机制。康贝特人将以 刺突蛋白与ACE 2相互作用导致CFTR丧失的机制。SA#2：定义 SARS-CoV-2刺突蛋白驱动ENaC和NFκB信号转导激活的机制。我们将 确定刺突诱导的CFTR蛋白丢失导致TRADD激活的机制。 依赖NFκB ENaC信号传导。SA #3：确定强心苷对细胞的保护机制 和COVID-19疾病的动物模型。我们将测试强心苷类药物是否能阻止天然SARS- 上皮细胞的CoV-2感染和COVID-19的救援仓鼠模型。. 新奇和意义：据我们所知，这是COVID-19气道炎症首次被 追踪到CFTR信号传导的抑制。同样，COVID-19患者也是CF携带者，只有一个 野生型CFTR基因和只有50%的CFTR功能，最近报道患有更严重的COVID-19 症状和更早的死亡比正常受试者与COVID-19。

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.