Cigarette smoke preferentially induces full length ACE2 exposure in primary human airway cells but does not alter susceptibility to SARS-CoV-2 infection
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Abstract
Cigarette smoking has multiple serious negative health consequences. However, the epidemiological relationship between cigarette smoking and SARS-CoV-2 infection is controversial; and the interaction between cigarette smoking, airway expression of the ACE2 receptor and the susceptibility of airway cells to infection is unclear. We exposed differentiated air-liquid interface cultures derived from primary human airway stem cells to cigarette smoke extract (CSE) and infected them with SARS-CoV-2. We found that CSE increased expression of full-length ACE2 (flACE2) but did not alter the expression of a Type I-interferon sensitive truncated ACE2 that lacks the capacity to bind SARS-CoV-2 or a panel of interferon-sensitive genes. Importantly, exposure to CSE did not increase viral infectivity despite the increase in flACE2. Our data are consistent with epidemiological data suggesting current smokers are not at excess risk of SARS-CoV-2 infection. This does not detract from public health messaging emphasising the excess risk of severe COVID-19 associated with smoking-related cardiopulmonary disease.
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SciScore for 10.1101/2021.09.08.459428: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Primary human bronchial epithelial cell (HBEC) culture and other cell line culture: Primary human bronchial epithelial cells (HBECs) derived from a non-smoking donor (Cat# CC-2540, male; Lonza; Donor 1) or derived directly from a patient at Cambridge University Hospitals NHS Trust (Research Ethics Committee Sex as a biological variable Cat# CCL-185, male) Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Permeabilised cells were pelleted, stained for 15 minutes at room temperature in 100 µL of sheep anti-SARS-CoV-2 nucleocapsid antibody (MRC-PPU, DA114) at a concentration of 0.7 … SciScore for 10.1101/2021.09.08.459428: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Primary human bronchial epithelial cell (HBEC) culture and other cell line culture: Primary human bronchial epithelial cells (HBECs) derived from a non-smoking donor (Cat# CC-2540, male; Lonza; Donor 1) or derived directly from a patient at Cambridge University Hospitals NHS Trust (Research Ethics Committee Sex as a biological variable Cat# CCL-185, male) Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Permeabilised cells were pelleted, stained for 15 minutes at room temperature in 100 µL of sheep anti-SARS-CoV-2 nucleocapsid antibody (MRC-PPU, DA114) at a concentration of 0.7 µg/mL, washed and incubated in 100 µL AF488 donkey anti-sheep (Jackson ImmunoResearch #713-545-147) at a concentration of 2 µg/mL for 15 minutes at room temperature. anti-SARS-CoV-2 nucleocapsid antibody ( MRC-PPU , DA114suggested: Noneanti-sheepsuggested: (Jackson ImmunoResearch Labs Cat# 713-545-147, RRID:AB_2340745)Primary antibodies; anti-ACE2 antibody was initially Abcam 228349 but was discontinued part-way through this study and was then replaced with 21115-1-AP (Proteintech); Acetylated tubulin (T7451; Sigma); Muc5AC (MA5-12178; Invitrogen), SARS-CoV / SARS-CoV-2 (COVID-19) spike antibody [1A9] (GTX632604; Genetex), SARS-CoV-2 (COVID-19 anti-ACE2suggested: None21115-1-AP ( Proteintech); Acetylated tubulinsuggested: NoneSARS-CoV-2suggested: NoneExperimental Models: Cell Lines Sentences Resources , Calu3 (ATCC; Cat# HTB-55, male) HEK293T (ATCC; Cat# CRL-3216, female) (A549-ACE2, HEK293T-ACE2) have been used as negative or positive controls. HEK293Tsuggested: ATCC Cat# CRL-3216, RRID:CVCL_0063)A549 and Calu-3 cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) and Eagle’s Minimal Essential Medium (EMEM) respectively, supplemented as specified above. A549suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)Calu-3suggested: BCRJ Cat# 0264, RRID:CVCL_0609)Viral titre was determined by 50% tissue culture infectious dose (TCID50) in Huh7-ACE2 cells. Huh7-ACE2suggested: NoneSoftware and Algorithms Sentences Resources Composite images were generated and analysed using Fiji. Fijisuggested: (Fiji, RRID:SCR_002285)Quantification and Statistical Analysis: Statistical analyses of mRNA expression assays and infection quantification data were performed using Prism 8 software (GraphPad Software). Prismsuggested: (PRISM, RRID:SCR_005375)GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
Identifier Status Title NCT04583410 Recruiting Efficacy of Nicotine in Preventing COVID-19 Infection Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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