Expression of ACE2—a Key SARS-CoV-2 Entry Factor—Is Not Increased in the Nasal Mucosa of People with Cystic Fibrosis

Mutations in CFTR (cystic fibrosis transmembrane regulator) lead indirectly to impaired innate defense of the respiratory tract, and people with cystic fibrosis (PwCF) develop a host of bacterial and fungal infections. The role of viruses in cystic fibrosis (CF) pulmonary disease has gained increased attention, and multiple studies have shown that viral infections are common antecedents for pulmonary exacerbations. Specifically, influenza, respiratory syncytial virus, and rhinovirus, all RNA viruses, are the most common and are sometimes associated with precipitous, unrecoverable loss of lung function in PwCF (1–5). Less well understood is whether PwCF are predisposed to developing specific viral infections or are more susceptible to severe complications once infected. The global coronavirus disease (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a highly contagious RNA virus that has caused millions of deaths worldwide. SARS-CoV-2 requires ACE2 (angiotensin-converting enzyme 2) and priming protease TMPRSS2 (transmembrane serine protease 2) for entry into epithelial cells, and studies published early in the pandemic have shown that inflammation, particularly IFN responses to viruses, can lead to increased ACE2 expression (6, 7). It has been demonstrated that ACE2 expression is highest in the nasal epithelium, which is a primary site of SARS-CoV-2 infection, and gradually decreases into the distal airways and parenchyma (8). As PwCF often have mucus accumulation and neutrophilic inflammation of the upper and lower airways, we wished to evaluate whether they have increased ACE2 nasal expression. Potentially mitigating this risk is the finding that chronic inflammation can also induce the expression of a short form of ACE2 that lacks a receptor-binding domain andmay lead to decreased susceptibility to infection (9). We therefore tested whether expression of ACE2 and the inflammatory response are different in the nasal epithelium of PwCF compared with healthy volunteers (HVs). We reanalyzed our previously published pilot and validation data sets, for which the study methods and demographics of the recruited cohort have already been reported (10). (Detailed code for analysis and reproducibility is available at https://github.com/ NUPulmonary/2021_CF_ACE2.)We separated PwCF into two cohorts, one consisting of PwCF who were F508del homozygotes and the second of those who were F508el compound heterozygotes. To test whetherACE2 and TMPRSS2 are differentially expressed, we first performed direct pairwise comparison in our pilot cohort, which consisted of six HVs and seven PwCF, and did not detect differentially expressed genes between PwCF and HVs (data not shown). In our larger validation cohort, we compared samples from HVs (11 subjects) and PwCF homozygous (13 subjects) and heterozygous (10 subjects) for F508del. We found 709 genes upregulated in HVs compared with 2,299 genes upregulated in PwCF homozygous for F508del (false discovery rate [FDR], q, 0.05) and 607 genes upregulated in HVs compared with 2,277 genes upregulated in PwCF compound heterozygous for F508del (FDR q, 0.05). Although ACE2was not among the differentially expressed genes in any comparison, expression of TMPRSS2was decreased in PwCF heterozygous for F508del but not in PwCF homozygous for F508del (Figure 1A; also see Figures E1B and E1C and Tables E1 and E2 in the data supplement). As gene expression profiles in bulk RNA-sequencing (RNA-seq) samples can be affected by the sample cell composition, we performed k-means clustering on the basis of highly variable genes (ANOVAlike test in DESeq2 [11]) and hierarchical clustering on subjects to better understand sources of variation in our data sets. This analysis identified three clusters (Figures 1B and E1A and Tables E3 and E4). We further investigated the larger data set and found that cluster A included HVs (11 of 11 subjects) and both homozygous (7 of 13 subjects) and compound heterozygous (3 of 10 subjects) PwCF. Subject cluster A was characterized by increased expression of genes related to secretory and ciliated epithelial cells, such as BPIFA1 (BPI fold containing family Amember 1), CAPS (calcyphosine), and TPPP3 (tubulin polymerization promoting protein family member 3) (Figure 1C). Subject clusters B and C contained both homozygous and compound heterozygous PwCF but no HVs and were characterized by genes restricted to neutrophils and genes involved in immune responses, such as CSF3R (colony stimulating factor 3 receptor), FCGR3B (Fc g receptor IIIb), and ITGA2 (integrin subunit a 2) (Figures 1B and 1C). As expected, ACE2was not among highly variable genes. TMPRSS2 belonged to gene cluster 1. To better understand the cellular composition of samples, we then performed in silico deconvolution of bulk RNA-seq signature using the AutoGeneS algorithm and publicly available single-cell RNA-seq data of nasal epithelium as a reference (12, 13). This analysis demonstrated that cluster A was dominated by an abundance of secretory and ciliated epithelial cells (Figure 1D; and Table E5). Cluster C in addition to epithelial cells also contained neutrophils, whereas cluster B was dominated by neutrophils. To ensure that the sample composition did not influence our analysis of ACE2 or TMPRS2 expression, we then performed a pairwise comparison using only samples from cluster A (epithelial cluster), which contained samples fromHVs (n=11) and PwCF homozygous (n=7) and heterozygous (n=3) for F508del. This analysis identified 379 genes upregulated in HVs and 146 genes upregulated in PwCF homozygous for F508del, and enrichment analysis for gene ontology biological processes demonstrated that these genes were related to T-cell activation (see Tables E6 and E7). When we performed a comparison between HVs and PwCF heterozygous for F508del with similar cell type composition, only 44 and 6 genes were upregulated in HVs and PwCF heterozygous for F508del, respectively (FDR, q, 0.05) (Figure 1E and Table E8). ACE2 and TMPRS2were not differentially expressed in either comparison of cluster A. A similar analysis in the pilot data set did not detect differential gene expression forACE2 and This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0. For commercial usage and reprints, please e-mail Diane Gern.