key: cord-0926010-utbhs9nj authors: Lu, Wenju; Liu, Xiaoqing; Wang, Tao; Liu, Fei; Zhu, Airu; Lin, Yongping; Luo, Jieping; Ye, Feng; He, Jianxing; Zhao, Jincun; Li, Yiming; Zhong, Nanshan title: Elevated MUC1 and MUC5AC mucin protein levels in airway mucus of critical ill COVID‐19 Patients date: 2020-08-10 journal: J Med Virol DOI: 10.1002/jmv.26406 sha: 430af3c9c346b704c05e37125400b15fdf29d24e doc_id: 926010 cord_uid: utbhs9nj Coronavirus disease 2019 (COVID‐19) patients exhibit a spectrum of respiratory symptoms like cough and dyspnea1‐3. Airway mucus is an adhesive viscoelastic gel composed mostly of high‐molecular weight mucous glycoproteins and water, which is important in maintaining lung function and health, pathological mucus hypersecretion may cause airway obstruction and lead to respiratory distress. Mucin (MUC) glycoproteins are the major macromolecular components of mucus, which are classified into two major types: the gel‐forming, secreted mucins (i.e, MUC5AC) and the membrane‐tethered mucins (i.e. MUC1)4. Here, with an attempt to understand the lung changes, we sought to provide a delineation of the components of airway mucus from COVID‐19 patients. This article is protected by copyright. All rights reserved. gel composed mostly of high-molecular weight mucous glycoproteins and water, which is important in maintaining lung function and health, pathological mucus hypersecretion may cause airway obstruction and lead to respiratory distress. Mucin (MUC) glycoproteins are the major macromolecular components of mucus, which are classified into two major types: the gel-forming, secreted mucins (i.e, MUC5AC) and the membrane-tethered mucins (i.e. MUC1) 4 . Here, with an attempt to understand the lung changes, we sought to provide a delineation of the components of airway mucus from COVID-19 patients. In order to clean airway obstruction, respiratory tract mucus was aspirated and collected via bronchoscopy from COVID-19 patients with critical illness, and optical coherence tomography (OCT) was applied via bronchoscopy to obtain cross-sectional images of the bronchiole. For healthy control, sputum was induced by inhaled hypertonic (3%) saline solution delivered with an ultrasonic nebulizer. After collection, sputum was processed as previously described for components analysis 5 . Medical history, clinical and laboratory data of the participants were extracted from electronic medical records. The study was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University (No. 2020-65). The requirement for informed consent was waived because the study was observational and the family members were in quarantine. MUC5AC (sc-21701, Santa Cruz, Shanghai, China) and MUC1 (sc-6827, Santa Cruz) in airway mucus were measured using enzyme-linked immunosorbent assay (ELISA), and MUC1-cytoplasmic tail (CT, clone EP1024Y, abcam, Shanghai, China) levels were measured by western blotting as described previously 5 . The levels of MUC5AC and MUC1 were normalized to their average signal reading of healthy control group. A total of 16 COVID-19 patients were included in this study ， the clinical characteristics of the recruited subjects were shown in Supplemental Table 1 . There was no significant deviation in distribution of age, or gender between the This article is protected by copyright. All rights reserved. cohorts of case and control subjects. All the COVID-19 patients were admitted to intensive care unit (ICU) because of low oxygenation index (199 ± 23 mmHg), and 79% of them received mechanical ventilation. Blood laboratory test showed elevated inflammatory indexes including leukocyte count, C reactive protein (CRP), and interleukin-6 (IL-6) in most of the COVID-19 patients (Supplemental Table 2 ). OCT indicated clear bronchiole in healthy controls ( Figure 1A ) and mucus retention in the bronchiole of COVID-19 patients ( Figure 1B) . A volume of 1~8 mL white to grey sputum with high viscosity ( Figure 1C ) was aspirated from the respiratory tract of 16 COVID-19 patients. Induced sputum from healthy control were clear and transparent with low viscosity. Compared to healthy control, airway mucus from COVID-19 patients had a higher level of MUC5AC ( Figure 1D ), MUC1 ( Figure 1E) , and MUC1-CT fragment ( Figure 1F ). However, there was no significant differences in the concentration of total protein, sodium or chloride in the airway mucus from COVID-19 patients when compared to healthy control (Supplemental Table 3 ). Although more than half of COVID-19 patients presented with dry cough 6 , our study provided direct evidence showing mucus retention in the small airway of COVID-19 patients, and patients were not able to expectorate by themselves and need bronchoscopy aspiration to help them to clean respiratory tract. The sputum from these COVID-19 patients was viscous, which is not surprising as MUC5AC levels are extremely high, hyperconcentration of this gel-forming mucin dehydrates airway surfaces and causes mucus adhesion, which may contribute to airflow obstruction and respiratory distress. Clearance of airway mucus is an important way to increase oxygen and carbon dioxide exchange, bronchoscopy aspiration of airway mucus was used in all our patients to relieve hypoxia. In our center, all the 16 critical ill COVID-19 patients recovered and were discharged from hospitalization, which may attribute to our aggressive clearance of respiratory tract. It is of note that bedside bronchoscopy may not be available in some hospitals as the medical resources are limited during the COVID-19 pandemic, carbocisteine has been reported to inhibit airway MUC5AC secretion, which could be used to reduce sputum viscosity and elasticity in COVID-19 patients. In addition, hydration of sputum by aerosolized hypertonic saline solutions or mannitol, and dilation of bronchi via aerosolized salbutamol may facilitate sputum expectoration. MUC1 is a membrane-tethered mucin expressed on the apical surface of epithelial cells 7, 8 . Since MUC1-CT fragment is on the cytoplasmic side of the cells, the elevated sputum CT fragment in COVID-19 patients could come from detached and disrupted epithelial cells, which is evidenced by the pathological findings of diffuse alveolar damage with fibromyxoid exudates and macrophage infiltration in the lung tissue from COVID-19 patients 9 . The limitation of our study is that induced sputum was used in the control group to compare airway mucus aspirated via bronchoscopy from COVID-19 patients, because it was very difficult for the COVID-19 patients to expectorate sputum even with hypertonic saline solution inhalation. The findings may suggest that increased level of mucins in the airway mucus may contribute to high viscosity of airway mucus and sputum retention in the small airway of COVID-19 patients, airway mucus clearance may be indicated to relieve respiratory distress, and MUC5AC may serve as a target for mucolytic agents in treating COVID-19. And MUC1-CT may serve as an indicator reflecting the severity of airway and alveolar epithelial cell damage. Xu Z, Shi L, Wang Y, et al. Pathological findings of COVID-19 associated with acute respiratory distress syndrome. The Lancet Respiratory medicine. 2020. From China: hope and lessons for COVID-19 control. The Lancet Infectious diseases Review of the Clinical Characteristics of Coronavirus Disease 2019 (COVID-19) Clinical characteristics and outcomes of hospitalised patients with COVID-19 treated in Hubei (epicenter) and outside Hubei (non-epicenter): A Nationwide Analysis of China. The European respiratory journal The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system Sputum mucin 1 is increased during the acute phase of chronic obstructive pulmonary disease exacerbation Clinical Characteristics of Coronavirus Disease 2019 in China. The New England journal of medicine Muc1 cell surface mucin attenuates epithelial inflammation in response to a common mucosal pathogen MUC1 mucin: a peacemaker in the lung This work was supported by grants from the National Key R&D Project The authors have declared that there is no conflict of interest.