Association of endopeptidases, involved in SARS‐CoV‐2 infection, with microbial aggravation in sputum of severe asthma

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S U PP O RTI N G I N FO R M ATI O N
Additional supporting information may be found online in the Supporting Information section. DOI: 10.1111/all.14731

Association of endopeptidases, involved in SARS-CoV-2 infection, with microbial aggravation in sputum of severe asthma
To the Editor, COVID-19 can be a serious multisystem disease caused by the SARS-CoV-2 coronavirus, and the current pandemic has affected more than 80 million people and caused nearly two million deaths worldwide. The SARS-CoV-2 virus attaches to angiotensin-converting enzyme 2 (ACE2) receptors on the host cell membrane, with the help of dipeptidyl peptidase 4 (DPP4), both exopeptidases. 1 Cleavage of the virus spike protein (S-protein) by endopeptidases, such as transmembrane protease, serine 2 (TMPRSS2) and furin, occurs following which the virus enters the host cell leading to virus replication. 1 Other enzymes, such as the sialyltransferases, ST6GAL1 and ST3GAL4, play a role for the synthesis of influenza A virus entry receptors 2 ; however, their role in SARS-CoV-2 infection has not been elucidated.
Asthma is a chronic inflammatory airway disease affecting 350 million people worldwide. It has not been linked to serious outcomes when presenting with COVID-19 infection, although a higher risk of death has been reported in severe asthma populations. 3 The heterogeneous inflammatory nature of asthma raises the possibility that the type of asthmatic inflammation might determine the outcome of SARS-CoV-2 infection in asthma. Type 2 (T2) inflammatory markers have been associated with decreased ACE2 expression in asthma 4,5 that could underlie the reduced risk of SARS-CoV-2 infection in asthmatics. In contrast, non-T2 asthma, particularly neutrophilic asthma, has been associated with higher ACE2 and endopeptidases (TMPRSS2 and furin) expression as compared with the T2-high phenotype 4,5 that might imply a worse outcome with COVID-19 infection.
Airway microbial imbalances have been reported in asthma, particularly in severe non-T2 asthma, and are characterized by decreased microbial α-diversity with increased pathogenic bacterial abundances in association with neutrophilia. 6 Endopeptidases involved in S-protein cleavage such as furin may also play a role in the cleavage of pathogenic bacteria such Streptococcus pneumoniae and Pseudomonas aeruginosa or bacterial toxins. 7,8 High expression of such endopeptidases may be associated not only with a higher risk of SARS-CoV-2 infection but also with microbial imbalances in severe asthma. Therefore, the aim of this study was to investigate associations of sputum endopeptidases gene expression with metagenomics composition and whether they could be used to stratify asthma patients according to risk of SARS-CoV-2 infection.
We examined the relation of SARS-CoV-2-associated endopeptidases with the airway bacterial composition, SARS-CoV-2associated exopeptidases and sialyltransferases, and inflammatory profile (cells and proteins) in 120 sputum samples collected from severe nonsmoking asthmatics, severe smoking asthmatics, mild-moderate asthmatics, and healthy controls of the Unbiased BIOmarkers in PREDiction of respiratory disease outcomes (U-BIOPRED) adult cohort. 9 Definition of asthma severity within the U-BIOPRED cohort has been presented in details elsewhere. 9 Sputum transcriptomics, SomaScan ® proteomics, and metagenomics were assayed as described previously. 5,6 Gene set variation analysis (GSVA) was is equal to zero. Subsequently, subjects were subdivided into two groups according to their ESs, that is, endopeptidase-high (ES >0, n = 60) and endopeptidase-low expression group (ES <0, n = 60).
These were compared according to sputum inflammatory markers, metagenomics α-diversity measures, and gene expression of the exopeptidases, ACE2, DPP4, and sialyltransferases (ST3GAL4 and ST6GAL1). The two groups were also compared with respect to current intake of antibiotics, oral corticosteroid (OCS), OCS normalized dosage (in mg), and history of hypertension and diabetes diagnoses. The differential bacterial abundance between endopeptidase groups was computed using edgeR after relative log expression normalization, while proteomics differential abundance was computed using limma. Pathway enrichment analysis of differentially abundant proteins in the endopeptidase-high group was performed using the Reactome database in g: Profiler (https://biit.cs.ut.ee/gprof iler/ gost).
Severe nonsmoking (n = 61) and smoking (n = 23) asthmatics showed the highest median expression ES of endopeptidase as compared to mild-moderate asthmatics (n = 20) and healthy controls (n = 16) ( Figure 1A), consistent with previous findings. 5 The endopeptidases ESs were significantly correlated with sputum neutrophil absolute counts (r s = 0.55, p = 7.7 × 10 −11 ) and percentages (r s = 0.58,  The present findings suggest that personalized therapies, such as those targeting neutrophils (eg, anti-IL-17), endopeptidase inhibitors (eg, neprilysin inhibitors), and/or antimicrobial compounds, might be tailored to asthma patients with high risk of SARS-CoV-2 infection.
In conclusion, these findings in sputum highlight that it is important to assess overall microbial profile in relation to SARS-CoV-2associated proteases in order to adequately assess risk of infection in patients with severe neutrophilic asthma.

ACK N OWLED G EM ENTS
We would like to thank all the patients who gave written and signed consent to take part in the U-BIOPRED study.