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.