Protein expression of <scp>SARS‐CoV</scp>‐2 receptors <scp>ACE2</scp> and <scp>TMPRSS2</scp> in allergic airways after allergen challenge

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes the angiotensin-converting enzyme 2 (ACE2) receptor in conjunction with the cell surface transmembrane protease serine 2 (TMPRSS2) to enter host cells.1 The type 2 (T2) cytokine IL-13 has been shown to downregulate ACE2 mRNA levels in cultured epithelial cells of asthmatic donors,2, 3 however, whether environmental triggers causing elevated airway IL-13 levels in vivo translates to changes in protein levels in asthmatic airways is unknown. By immunofluorescence microscopy, we assessed protein expression of ACE2 and TMPRSS2 under T2 high conditions after allergen challenge of the upper and lower airways of subjects with allergic asthmatics and allergic rhinitis, respectively (Table S1 Supporting Information). The study was approved by the Hamilton Integrated Research Ethics Board, and participants provided informed written consent. Eleven participants with mild allergic asthma (FEV1 ≥ 70% predicted, methacholine PC20 ≤ 16 mg/mL, skin prick test positive) underwent whole lung allergen inhalation challenges (AIC), resulting in early and late bronchoconstriction responses, sputum eosinophilia (Figure 1A,B), and increased sputum eotaxin-1, eosinophil-derived neurotoxin (EDN) and the T2 cytokines IL-5, and IL-13 post-challenge (p < .05) (Table S2 supplementary material). Ten participants underwent a second AIC using the same dose of allergen, and endobronchial biopsies were obtained before and again at 24 h post-AIC. There was a significant reduction in the number of bronchial tissue cells immuno-positive for ACE2, TMPRSS2, and double positive for ACE2/TMPRSS2 (p = .002, p = .014, p = .002, respectively) measured 24 h post-AIC (Figure 1C,D). There was no correlation between SARS-CoV-2 receptor immuno-positive cells and levels of biomarkers in sputum. Ten allergic asthmatics with co-morbid allergic rhinitis completed a crossover study with nasal allergen challenge (NAC) conducted after 21 days of intranasal placebo or triamcinolone (220 mcg BID) treatment. The NAC-induced changes in peak nasal inspiratory flow rate, nasal lavage eosinophils (Figure 2A,B) IL-5, IL-13, and eotaxin-1 (Table S2 Supporting Information) observed during placebo treatment were all significantly attenuated by triamcinolone (p < .05). In biopsies of inferior nasal turbinate, NAC did not change the number of cells immuno-positive for ACE2 or TMPRSS2 (Figure 2C,D), and we did not observe a relationship between immunopositivity for ACE2, TMPRSS2, and biomarkers in nasal lavage. Previous studies have reported that nasal and bronchial allergen challenges lower ACE2 mRNA transcript in epithelium of nasal and bronchial brushing, respectively.3 Using in vitro model data sourced from Gene Expression Omnibus, Jackson et al found that IL-13 reduced ACE2 mRNA expression in differentiated nasal and bronchial epithelium. Using cultured primary human bronchial epithelial cells Stocker et al reported that IL-13 decreases long ACE2 mRNA isoforms and reduces glycosylation of full length ACE2 protein, thereby limiting expression on the apical side of ciliated cells exposed to viral infection.2 We therefore hypothesized that elevation of IL-13 levels after allergen challenge and lowering of IL-13 with corticosteroid treatment would correspondingly regulate ACE2 protein expression in airways. Indeed, ACE2 and TMPRSS2 immunopositivity was significantly reduced in bronchial tissue after AIC. In nasal tissue, however, interpretation of the data is inconclusive due to low ACE2 and TMPRSS2 protein levels measured at baseline in inferior nasal turbinate tissue, and by the small study sample size. The proposed protective mechanisms of IL-13 raise the possibility that T2 high airways may be protective against SARS-CoV-2, but to date, this has not been supported by clinical observations. In general populations, there is no clear association between asthma and SARS-CoV-2 infectivity or hospitalization.4, 5 Counterintuitive to a proposed protective role of IL-13, treatment with dupilumab, a monoclonal antibody that blocks IL-13 signaling, was reported to improve survival in asthmatic patients compared with matched controls after SARS-CoV-2 infection.6 Collectively, these data suggest that despite dampening of ACE2 and TMPRSS2 receptor expression in airways by IL-13, there are other factors contributing meaningfully to the rate of SARS-CoV-2 infectivity and hospitalization in asthmatic patients. All authors contributed to the study design, acquisition or analysis of data, were involved with drafting of this manuscript, and approved the final version and are accountable for all aspects of the work. We would like to acknowledge funding from AstraZeneca Canada (ESR 20-20723) and Mitacs (IT22844). The authors have no conflict of interest related to this manuscript. Outside of this work, MD reports research funding from Gilead and Janssen; DDS reports Advisory Board, research funding, speaking fees from GSK, Sanofi, Stryker; PMB reports personal fees for consulting or speaker fees from AstraZeneca, GSK, MedImmune, Chiesi, Menarini and Covis and research grants from AstraZeneca, MedImmune, Biohaven, Merck and Bayer; GMG reports personal fees for consulting or speaker fees from AstraZeneca, Sanofi–Regeneron and research grants from Biohaven, Genentech, BioGaia, Novartis. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Data S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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