Traffic-derived particulate matter and angiotensin-converting enzyme 2 expression in human airway epithelial cells

Abstract

Background

The mechanism for the association between traffic-derived particulate matter less than 10 microns (PM ₁₀ ) and cases of COVID-19 disease reported in epidemiological studies is unknown. To infect cells, the spike protein of SARS-CoV-2 interacts with angiotensin-converting enzyme 2 (ACE2) on host airway cells. Increased ACE2 expression in lower airway cells in active smokers, suggests a potential mechanism whereby PM ₁₀ increases vulnerability to COVID-19 disease.

Objective

To assess the effect of traffic-derived PM ₁₀ on human airway epithelial cell ACE2 expression in vitro .

Methods

PM ₁₀ was collected from Marylebone Road (London) using a kerbside impactor. A549 and human primary nasal epithelial cells were cultured with PM ₁₀ for 2 h, and ACE2 expression (median fluorescent intensity; MFI) assessed by flow cytometry. We included cigarette smoke extract as a putative positive control. Data were analysed by either Mann-Whitney test, or Kruskal-Wallis with Dunn’s multiple comparisons test.

Results

PM ₁₀ at 10 μg/mL, and 20 μg/mL increased ACE2 expression in A549 cells (P<0.05, 0.01 vs. medium control, respectively). Experiments using a single PM ₁₀ concentration (10 μg/mL), found increased ACE2 expression in both A549 cells (control vs. PM ₁₀ , median (IQR) MFI; 470 (0.1 to 1114) vs 6217 (5071 to 8506), P<0.01), and in human primary epithelial cells (0 (0 to 591) vs. 4000 (2610 to 7853), P<0.05). Culture of A549 cells with 5% cigarette smoke extract increased ACE2 expression (n=4, 0 (0 to 28) vs. 9088 (7557 to 15831, P<0.05).

Conclusion

Traffic-related PM ₁₀ increases the expression of the receptor for SARS-CoV-2 in human respiratory epithelial cells.

SciScore for 10.1101/2020.05.15.097501: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Cells were washed twice and stained with either anti-ACE2 (Abcam, UK – ab189168, ab272690) or isotype control primary antibodies (Abcam, Ab171870), for 1 h at room temperature.	anti-ACE2 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Airway cells: The human alveolar type II epithelial cell line A549 was purchased from Sigma-Aldrich (Poole, UK) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with fetal bovine serum …

SciScore for 10.1101/2020.05.15.097501: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Cells were washed twice and stained with either anti-ACE2 (Abcam, UK – ab189168, ab272690) or isotype control primary antibodies (Abcam, Ab171870), for 1 h at room temperature.	anti-ACE2 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Airway cells: The human alveolar type II epithelial cell line A549 was purchased from Sigma-Aldrich (Poole, UK) and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) and penicillin-streptomycin (Lonza, Basel, Switzerland).	A549 suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
Software and Algorithms
Sentences	Resources
Human primary nasal epithelial cells were obtained from a non-smoking, non-vaping, healthy adult donor using a dental brush, maintained in airway epithelial cell growth medium (AECGM), with supplement kit (PromoCell®, Heidelberg, Germany) with Primocin (InvivoGen, San Diego, USA), and stored at passage 1 cryogenically in freezing media (AECGM: 10% FBS, 10% DMSO).	PromoCell® suggested: None
Analysis were performed using Prism 8 (GraphPad Software Inc.,	GraphPad suggested: (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: We detected the following sentences addressing limitations in the study:

There are limitations to this study. First, we did not determine whether increased ACE2 expression increases infection of airway cells with SARS-CoV-2. However, evidence for this is provided by reports of; i) an association between smoking and severity to COVID-19 (14) (15), and ii) increased expression of airway epithelial ACE2 in current smokers compared with never smokers (12). By contrast, Jackson et al (9) speculated that lower ACE2 mRNA expression in airway brush samples from children with allergic asthma decreases their susceptibility to SARS-CoV-2 infection. Second, we have not identified how PM10 upregulates ACE2 expression on airway cells. We have previously reported that PM-induced oxidative stress increases PAFR expression on primary bronchial epithelial cells (16), but the role of oxidative stress on ACE2 expression is as yet unknown. Finally, although the concentration of PM10 used in the present study is lower than that used in our previous in vitro study of PM and pneumococcal infection of airway cells (16), it remains unclear to what extent 10 μg/mL reflects in vivo exposure. Assessment of ACE2 expression in nasal brushings from individuals changing from low to high pollution exposure, for example during and after the COVID-19 lockdown, offers a way of non-invasively validating results from in vitro studies. In conclusion, this study provides the first mechanistic evidence that traffic-derived air pollution increases ACE2 expression in human airway cells and ...

Results from TrialIdentifier: No clinical trial numbers were referenced.

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:

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No funding statement was detected.
No protocol registration statement was detected.

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