Discussion
By analysing the transcriptome of three lower airway compartments (bronchial biopsies, brushings, and sputum cells), we provide evidence for the over-expression of the factors that may determine the entry and activity of the SARS-CoV2 virus into host cells, particularly ACE2 and FURIN in sputum cells of severe asthmatics, compared to the mild-moderate asthmatics or healthy controls. This is in contrast to reports of no differences in the expression of ACE2, TMPRSS2 and FURIN between mild and severe asthma and healthy controls in sputum or in airway epithelial brushings and biopsies (18, 19). Multivariate analysis showed that male gender and OCS use was associated with ACE2 expression in sputum, and male gender and BMI with TMPRSS2 and male gender with FURIN expression in bronchial brushings. Interestingly, male gender and obesity and also severe asthmatics who have had a recent course of OCS have been linked to a greater risk of death from SARS-CoV2 infection (6, 20, 21). Multivariate linear regression analysis also showed that the transcript levels of FURIN in sputum was significantly associated with severe asthma and sputum neutrophilia. Thus, patients with severe neutrophilic asthma, but not those with mild-moderate disease, may have the pathophysiological makeup that enhances the risk of severe infection by SARS-CoV-2. Our findings also indicate that the neutrophilic severe asthma may be more prone to a poorer outcome with SARS-CoV2. This finding is further supported by our associative analysis by molecular phenotype. Sputum ACE2 and FURIN transcripts were elevated in TAC2, an asthma endotype characterized by sputum neutrophilic inflammation and inflammasome activation signature.
The specific molecular cluster of TAC2 has been previously described in the U-BIOPRED cohort and is characterized by predominant sputum neutrophilic inflammation, inflammasome activation and neutrophilic activation(15). In the present study, there was significantly higher expression of FURIN in sputum of TAC2, compared to TAC1 and TAC3. Furthermore, FURIN expression levels in sputum correlated highly with the expression of 3 gene signatures associated with neutrophil activation signature, inflammasome activation signature, and also a signature that reflected an IL-6-trans-signalling pathway that we have also previously reported (22). The relationship between FURIN and neutrophils in sputum remained significant when adjusted for asthma severity, post-bronchodilator FEV1 (% predicted), or oral corticosteroid use. We also found modest but significant correlations between FURIN expression in bronchial brushings and bronchial biopsy and the expression of the neutrophil activation and IL-6 trans-signalling signature, and between FURIN expression in bronchial biopsy with the inflammasome signature. These findings lay emphasis on a potential link of FURIN with neutrophil activation, inflammasome, and IL-6 activation pathways, supported by high expression levels of IL-6 and neutrophil activation from lung epithelial cells infected with SARS-CoV2 in vitro (23) and by serum levels of IL-6 being a strong predictor for respiratory failure in severe COVID-19 infection (24). FURIN levels were not evaluated in the recent Severe Asthma Research Program-3 cohort analysis (19). The study by Bradding et al. similarly found no significant difference of FURIN levels in airway epithelial brushing and biopsy by asthma presence or severity, but it did not examine sputum samples (18). Interestingly, FURIN is most highly expressed in granulocytes such as neutrophils (https://www.proteinatlas.org/ENSG00000140564-FURIN/blood ). One could speculate that the presence of neutrophils in the airway mucosa could enhance the degradation of the spike protein on SARS-CoV-2 and facilitate viral entry into airway and inflammatory cells by FURIN (6). In addition, neutrophils in the airways may be an important site for the propagation of SARS-CoV2 down the airways to the respiratory epithelium, that could lead to the development of pneumonia, a process that leads to hypoxaemia and more severe disease.
We found increased expression of ACE2 in sputum cells of the TAC1 eosinophilic phenotype that had an enrichment of gene signatures for IL-13/ Th2 inflammation and of the TAC2 phenotype, compared to TAC3, a pauci-granulocytic phenotype with increased metabolic and mitochondrial function genes(15). ACE2 and TMPRSS2 expression in sputum were correlated with expression levels of the IL-13-Th2 signature. In bronchial biopsies, the levels of the 3 genes were all modestly correlated with that signature. On the other hand, atopy and sputum eosinophil counts were not significantly correlated with ACE2, TMPRSS2 and FURIN transcripts. We therefore have not confirmed the reports that ACE2 expression was reduced in mild-moderate asthmatics with T2-high compared to T2-low (25), and that ACE2 gene expression is positively correlated with Th2 gene expression in a group of asthmatics from mild to severe asthma (18).
One of the foremost reason for these different results is that the U-BIOPRED cohort represents a large proportion of patients with very severe asthma as reflected by the large number of frequent exacerbations and the degree of airflow obstruction and the nearly 50% of patients on oral corticosteroid therapy (14), when compared to the recent study of severe asthmatics that did show any differences in expression of these 3 SARS-CoV2 entry genes (18). Thus, there may have been due to confounding by therapy (such as the use of OCS which was more highly prevalent in U-BIOPRED participants), the nature of the underlying T2 inflammatory process as well as the different but more comprehensive analytical approach taken in our study. Also, there may be differences in the asthma-driving mechanisms whereby the presence of differential contributions of Th2, Th17 and Th1 pathways in each asthmatic individual (26, 27), may determine the overall expression of the SARS-CoV-2 entry and activation genes.
Olfactory and taste disturbance is a common symptom of COVID-19, and the nasal epithelium is suggested to be a major route of viral infection (28, 29). The gene expression levels of ACE2, TMPRSS2 and FURIN nasal brushings in our patients did not differ by asthma severity or molecular phenotype (data not presented), and also importantly, the levels were not significantly different in the presence of co-morbid nasal polyps (Supplementary Fig S2) . However, the absolute gene expression levels of ACE2 and TMPRSS2 were higher in nasal brushings than in lower airway compartments (Supplementary Table S3) , supporting a recent report that the nasal epithelium is a major site of infection (29). Based on the expression patterns in the upper and lower airways, we suggest that severe asthmatics might have a higher risk of poorer outcomes from COVID-19, although the risk of infection via the nasal route is unlikely to be different by asthma severity.
Our study has major limitations. First, this is a cross-sectional study and could not determine causal relationships. Several features of severe asthma, such as sputum eosinophils and neutrophils, lung function or OCS use may be inter-related, but we used multivariate analyses to address this. Second, we did not find any positive correlations between smoking status (ever versus never-smokers) and target gene expressions, despite the fact that smoking can increase the expression of ACE2 in the airways (30-32). However, it is likely that there were not enough current smokers in the severe asthma group (6.7%) to show any difference. Third, our analyses were only performed at gene expression levels, which should be confirmed at protein levels. On the other hand, the strength of our analysis is that it evaluated expression in a range of airway cell populations (epithelial brushing, bronchial biopsy and sputum cells), allowing an assessment of the molecular phenotypes. U-BIOPRED also had a large sample size of well-characterised severe asthma participants that was recruited at the same time as the healthy controls and mild-moderate asthmatics using the same pre-defined protocol. Finally, we have not examined protein expression of these SARS-CoV2 entry factors and particularly the protease activity of TMPRSS2 and FURIN.
In conclusion, we found higher gene expression levels of ACE2 and FURIN in sputum of severe asthma compared to those of non-severe asthma. Sputum FURIN levels highly correlated with sputum neutrophils and were higher in an asthma endotype characterized by sputum neutrophilia and inflammasome activation signature. Our data also supports the notion that the airway neutrophil may be a site of or potentiate the invasion by the SARS-CoV2 virus, thus increasing virus load and replication in patients with sputum neutrophilia. More importantly, the data also indicates that the severe neutrophilic asthma may be at risk of a poorer outcome if infected with SARS-CoV2 through the upregulation of FURIN, involved in the degradation of the SARS spike protein.