key: cord-284053-tna7e9dw
authors: Kimura, Hiroki; Francisco, Dave; Conway, Michelle; Martinez, Fernando D.; Vercelli, Donata; Polverino, Francesca; Billheimer, Dean; Kraft, Monica
title: Type 2 Inflammation Modulates ACE2 and TMPRSS2 in Airway Epithelial Cells
date: 2020-05-15
journal: J Allergy Clin Immunol
DOI: 10.1016/j.jaci.2020.05.004
sha: 
doc_id: 284053
cord_uid: tna7e9dw

Abstract Background SARS-CoV-2 has dramatically changed our world, country, communities and families. There is controversy regarding risk factors for severe COVID-19 disease. It has been suggested that asthma and allergy are not highly represented as co-morbid conditions associated with COVID-19. Objective To extend our work in interleukin (IL)-13 biology to determine if airway epithelial cell expression of two key mediators critical for SARS-CoV-2 infection, angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2) are modulated by IL-13. Methods We determined effects of IL-13 treatment on ACE2 and TMPRSS2 expression ex vivo in primary airway epithelial cells from participants with and without type 2 asthma obtained by bronchoscopy. We also examined expression of ACE2 and TMPRSS2 in two datasets containing gene expression data from nasal and airway epithelial cells from children and adults with asthma and allergic rhinitis. Results IL-13 significantly reduced ACE2 and increased TMPRSS2 expression ex vivo in airway epithelial cells. In two independent datasets, ACE2 expression was significantly reduced and TMPRSS2 was significantly increased in the nasal and airway epithelial cells in type 2 asthma and allergic rhinitis. ACE2 expression was significantly negatively associated with type 2 cytokines while TMPRSS2 expression was significantly positively associated with type 2 cytokines. Conclusion IL-13 modulates ACE2 and TMPRSS2 expression in airway epithelial cells in asthma. This deserves further study with regard to any effects asthma and atopy may render in the setting of COVID-19 infection.

Since its recognition in December 2019, the outbreak of COVID-19 caused by SARS-91

CoV-2 infection has generated strong concern among individuals presenting with 92 underlying medical conditions. Individuals with chronic lung diseases or asthma are at 93 higher risk for developing severe complications from COVID-19 (1). Interestingly, 94 among patients with allergy and asthma, there is controversy as to whether these 95 represent comorbid conditions that increase risk for COVID-19. One study that reported 96 140 cases of COVID-19 in Wuhan, China, indicated no self-reported cases of asthma, 97 allergic rhinitis, atopic dermatitis, and food allergy among infected patients (2). 98

Moreover, a report by Dong and colleagues suggested a low prevalence of asthma and 99 allergy in pediatric cases, and also found a low prevalence of rhinitis and atopic 100

infection globally and reported a low prevalence of co-existing respiratory disease 102 overall in patients with acute COVID-19 infection (4). However, a recent report from 103 Seattle, Washington describing 24 cases of acute lung injury associated with COVID-19 104 described 3 of 24 patients who had asthma and had been treated with oral 105 corticosteroids within one week of presenting due to an asthma exacerbation (5). A 106 recent publication from COVID-19-Associated Hospitalization Surveillance Network 107 (COVID-NET) describing 180 patients admitted to hospitals in the United States from 108

March 1-22, 2020 reported that 17% carried the diagnosis of asthma (6). However, 109 information regarding severity, medication requirements and co-morbid conditions of 110 these patients are not currently known. Therefore, the controversy exists, as it is well 111

known that viral infections exacerbate asthma (7).

For effective host cell entry, SARS-CoV-2 relies on two critical proteins, 113 angiotensin-converting enzyme 2 (ACE2) (8) and transmembrane serine, protease 2 114 (TMPRSS2) (9). Here, we demonstrate that interleukin (IL)-13, a cytokine associated 115 with type 2 (T2) asthma, suppresses ACE2 receptor expression and significantly 116 increases TMPRSS2 expression in airway epithelial cells from participants with T2 117 asthma and atopy. Based on these findings, we hypothesized that T2 cytokines 118 modulate ACE2 and TMPRSS2 expression in the airway epithelial cell in asthma and 119 atopy. 120

Participants were recruited from the population in Tucson, Arizona and the 123 surrounding areas. Informed consent was obtained from each participant (18-65 years 124 of age). Asthmatic participants met GINA criteria for mild and moderate asthma 125

including the presence of reversibility of airflow obstruction or airways responsiveness 126 with a provocative concentration of methacholine resulting in a 20% fall in FEV 1 (PC20 127 FEV 1 ) of ≤ 8 mg/ml or < 16 mg/ml if they were taking inhaled corticosteroids (10). The 128 presence of atopy was determined using skin testing; peripheral eosinophils and 129 fractional exhaled nitric oxide (FeNO) was measured. Healthy participants had no 130 evidence of airflow obstruction, and no history of pulmonary disease; the presence of 131 atopy was not an exclusion. Exclusion criteria included an exacerbation of asthma 132 within four weeks of study requiring antibiotics and/or corticosteroids, greater than 10-133 pack year history of tobacco use or any cigarette use in the last year and any other 134 significant medical conditions. 135 136

Participants underwent bronchoscopy with endobronchial-protected brushing as 138 previously described (11). The brushing of the proximal airways to obtain bronchial 139 epithelial cells was performed under direct visualization using a separate protected 140 cytologic brush for each pass, for a total of eight passes. Participants were discharged 141 when their forced expiratory volume in one second (FEV 1 ) achieved 90% of their pre-142 bronchoscopy, post-albuterol value. 143

Freshly isolated airway bronchial epithelial cells from endobronchial brushings 146

were cultured with PneumaCult-EX Plus (StemCell Technologies, Vancouver, BC, 147 Canada). After reaching confluence, cells were trypsinized and seeded onto collagen-148 coated polyester Transwell ® insert membranes of 12-mm diameter (Corning, Lowell, 149 MA, USA), at a concentration of 5 × 10 4 /well. The cells were then cultured at air-liquid 150 interface (ALI) using PneumaCult-ALI (StemCell Technologies) and allowed to 151 differentiate for 2 weeks as previously described (11). Cells were stimulated with 10 152 ng/ml IL-13 (Peprotech, Rocky Hill, NJ, USA) for 48 hours. Epithelial cells were 153 collected for RT-PCR analysis of ACE2 and TMPRSS2 (see PCR methods below). 154

Conditions were performed in triplicate with appropriate unexposed controls. 155 156

Total mRNA was extracted according to RNeasy Plus Mini Kit instructions (Qiagen) and 158 cDNA was synthesized using 1 µg of RNA (Applied Biosystems, Foster City, CA, USA). 159

Real-time PCR was performed on a CFX96 Touch™ (Bio-Rad, Hercules, CA, USA) 160

using TaqMan™ probes (Applied Biosystems) specific for PPIA, ACE2, and TMPRSS2. 161 The data were presented per the 2 -∆∆Ct once analysis was complete. We used linear 203 mixed effect models to evaluate IL-13 treatment and asthma status on ∆Ct values. The 204 mixed model accommodates the nesting structure of participants within asthma group, 205 and treatment replicates for each participant. 206

For analysis of the public databases, differences among the groups were 207 analyzed using Wilcoxon rank sum test or Steel's multiple comparison test, as 208 appropriate. The trend among the groups was analyzed using the Jonckheere-Terpstra 209 test. In categorizing T2 low and T2 high groups in GSE19187 and GSE4302 datasets 210 respectively, unsupervised hierarchical clustering including values of three T2 signature genes, calcium-activated chloride channel regulator 1 (CLCA1), periostin (POSTN), and 212 serpin peptidase inhibitor, clade B, member 2 (SERPINB2) using the Euclidean metric 213 with complete linkage was performed, as previously described (17). Correlations 214 between nonparametric data were undertaken using Spearman's rank correlation. 215

Statistical significance was defined as p<0.05. All analyses were performed using R 216 version 3.6.1. software (The R Foundation; http://www.r-project.org/) or GraphPad 217

Results 219

Participant demographics for the ex vivo study are shown in Table 1 . The 221 asthmatic participants were mild to moderate in severity based upon medication 222 requirements with one patient requiring controller medication. Asthmatic participants 223 exhibited good to fair asthma control. All asthma and non-asthma participants had a 224 history of atopy based upon their clinical history, presence of allergic rhinitis, and 225 elevated FeNO. Total and allergen specific IgE were not available for these participants. 226 IL-13 reduces ACE2 and increased TMPRSS2 expression in airway epithelial 227 cells from both the asthma and non-asthma atopic groups (Fig 1) . As it been suggested 228 that ciclesonide may possess anti-viral properties (20), we excluded the one asthmatic 229 participant who used inhaled corticosteroids and repeated the analysis. We did not find 230 significant differences in ACE2 and TMPRSS2 expression (data not shown). In study GSE19187, nasal epithelial cells of participants with asthma and/or 240 allergic rhinitis demonstrate lower ACE2 expression compared to healthy participants 241 (Fig 2A) . Assessment of ACE2 expression by severity of symptoms (healthy, rhinitis 242 without asthma, rhinitis with controlled asthma, and rhinitis with uncontrolled asthma) 243

shows that participants with rhinitis and uncontrolled asthma (UA) demonstrate the 244 lowest nasal epithelial cell ACE2 expression (the Jonckheere-Terpstra trend test, Fig  245   2B ). In addition, participants who are defined as T2 high based upon three gene 246 signatures (CLCA1, POSTN, SERPINB2) (17) also demonstrate lower nasal epithelial 247 ACE2 expression compared to the T2 low and healthy control groups (Fig 2C) . 248

We next examined the association of nasal epithelial cell ACE2 expression with 249 T2 cytokines. IL-13, but not IL-4 and IL-5 significantly negatively correlates with ACE2 250 expression (Fig 3 A-C) . T2-driven genes CLCA1, POSTN and SERPINB2 show an 251 inverse correlation with ACE2 expression (Fig E1) . 252 253

In dataset GSE4302 containing airway epithelial cell mRNA, ACE2 expression is 255 lower (albeit not significantly) in asthmatic participants compared to control participants 256 (p=0.097) (Fig 4A) . When asthmatic participants are grouped into T2 high and T2 low 257 based upon the three gene signature expressions (17), the T2 high participants 258 demonstrate lower expression of ACE2 compared to healthy participants while T2 low 259 participants demonstrate similar expression as compared to healthy participants (Fig  260   4B) . 261

and IL-13 in asthmatic participants (Fig 5A-C) . Furthermore, two T2 cytokine-induced 263 genes (i.e., CLCA1 and SERPINB2, but not POSTN) significantly negatively correlate 264 with ACE2 expression when asthma and healthy groups are combined (see 265 supplement, Fig E2) . 266

In dataset GSE19187, participants with asthma and/or allergic rhinitis 269 demonstrate increased nasal epithelial cell expression of TMPRSS2 compared to 270 healthy participants whether combined (Fig 6A) or evaluated as separate groups (Fig  271   6B) . Moreover, the T2 high participants demonstrate significantly higher expression of 272 TMPRSS2 while the expression by T2 low participants is similar to healthy participants 273 ( Fig 6C) . There was a significant correlation between nasal epithelial TMPRSS2 gene 274 expression with CLCA1, POSTN and SERPINB2, but not with IL-4, IL-5 or IL-13 (Fig  275   E3) . While gene expression of TMPRSS2 is not different between healthy and 280 asthmatic participants (Fig 7A) , TMPRSS2 expression is higher (albeit not significantly) 281 in T2 high asthmatic compared to healthy participants (p=0.086). There is no difference 282 in TMPRSS2 expression between healthy and T2 low asthma (p=0.84) (Fig 7B) . 283

In addition, a significant correlation between airway epithelial TMPRSS2 and IL-4 284 expression is present when both groups are combined as well as for asthma only (Fig  285   E4 ). This relationship is not demonstrated for IL-5 and IL-13 (Fig E4) . Airway epithelial 286 cell TMPRSS2 expression significantly positively correlates with expression of CLCA1 and SERPINB2, but not POSTN when asthma and control groups are combined (Fig  288   E5) . 289

To our knowledge, this is the first report to show that IL-13 decreases ACE2 and 291 increases TMPRSS2 expression in T2 high asthma and in allergic rhinitis without 292 asthma. These ex vivo observations are supported by interrogation of two public 293 databases which include children and adults and in which we show that ACE2 294 expression is reduced and TMPRSS2 expression is increased in T2 asthma and rhinitis. 295

In addition, ACE2 and TMPRSS2 expression correlate with T2 cytokines, although in 296 opposite ways: ACE2 inversely correlates with T2 or T2 driven genes, while TMPRSS2 297 expression positively correlates with T2 or T2-driven genes. These analyses are 298 exploratory and provide a direction for future investigation. 299

The role of ACE2 in asthma is just beginning to be elucidated. The renin-300 angiotensin system (RAS) is a critical component in regulating multiple tissue and organ 301 functions, such as those of the cardiovascular system, kidney, lung, and liver, 302 specifically by maintaining homeostasis of blood pressure, electrolyte balance, and 303 inflammatory responses (21, 22). Renin, a protease produced predominantly in the 304 kidneys, cleaves angiotensinogen to generate angiotensin I (Ang I). Subsequently, 305 angiotensin-converting enzyme (ACE) cleaves Ang I to produce Ang II. Ang II is 306 hydrolyzed by various angiotensinases, such as ACE2. ACE2 is a terminal 307 carboxypeptidase and a type I transmembrane glycoprotein and a potent negative 308 regulator of the RAS. The imbalance in enzymatic activity of ACE/ACE2 has been 309 suggested to involve in pathogenesis in several diseases, including lung diseases (23). 310

In asthma, ACE2 may have a role due to its anti-inflammatory nature and ability 311 to inactivate Ang II and activate Ang1-7, the two counteractive systems strongly related to asthma. In an ovalbumin-challenged mouse model of T2 asthma, Ang1-7 modulated 313 ovalbumin-induced increases in total cell counts, eosinophils, lymphocytes, and 314 Whether inhaled or nasal corticosteroids are protective against SARS-CoV-2 358 infection merits comment, as it has been suggested that ciclesonide may possess anti-359 viral properties due to its ability to block viral replication (20). In our ex vivo study, one of 360 our asthmatic participants used inhaled corticosteroids. We excluded this participant 361 and repeated the analysis. We did not find significant differences in ACE2 and 362 TMPRSS2 expression (data not shown). Participants whose nasal airway epithelial cell 363 data were included in dataset GSE19187 did not receive treatment with nasal 364 corticosteroids for at least one month prior to enrollment. Similarly, participants whose 365 airway epithelial cells are included in dataset GSE4302 did not take inhaled or oral 366 corticosteroids for one month prior to enrollment. Thus, the association of ACE2 and 367 TMPRSS2 gene expression with T2 cytokines, atopy and T2 high asthma are not 368 dependent upon the effect of corticosteroids in this analysis. This issue should be 369 addressed in future investigation. 370

Our study has several strengths: Using airway epithelial cells from well-371 phenotyped atopic, non-asthma and asthmatic participants from our own laboratory, we 372 have shown that IL-13: 1) suppresses ACE2 gene expression and 2) increases 373 TMPRSS2 expression. We have observed similar results from two distinct datasets in 374 children and adults with rhinitis and asthma. This approach adds robustness to the 375 observation that IL-13 modulates the receptor system for COVID-19. Recently, Jackson 376 et al. has reported that underlying allergy and allergen exposure reduces the expression 377 of ACE2 (38) . Our data extends their findings by demonstrating 1) contrasting changes 378 in TMPRSS2 in response to IL-13 stimulation ex vivo 2) the effect of underlying allergy 379 was T2-specific, using unsupervised hierarchical clustering including T2-induced genes.

Limitations of our study included: 1) our sample size for the ex vivo studies is 381 small 2) data from publicly available datasets do not include detailed clinical information. 382

In summary, we provide evidence that T2 inflammation suppresses expression of 383 ACE2 and increases expression of TMPRSS2 in nasal and airway epithelial cells in 384 asthma and atopy. These observations may provide a foundation to elucidate the 385 relative role of these two mediators in cell entry and how T2 cytokines modulate 386 susceptibility to COVID-19. 387

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The authors would like to acknowledge Pascal Barbry, Ph.D. and Prescott Woodruff, 389 M.D. for use of their data in databases GSE19187 and GSE4302, respectively. The 390 authors would also like to acknowledge Ronald Schunk, R.T. for his assistance with 391 participant recruiting and bronchoscopy and Bianca Sierra for her assistance with ex 392 vivo experiments.

of IL-4, IL-5, and IL-13 with ACE2 in the airway epithelial cells from asthmatic 557 participants and when asthma and healthy groups are combined. r s : Spearman's rank 558 correlation coefficient. 559