key: cord-267536-rvhwl4ea authors: Miyashita, L; Foley, G; Semple, S; Grigg, J title: Traffic-derived particulate matter and angiotensin-converting enzyme 2 expression in human airway epithelial cells date: 2020-05-27 journal: bioRxiv DOI: 10.1101/2020.05.15.097501 sha: doc_id: 267536 cord_uid: rvhwl4ea Background The mechanism for the association between traffic-derived particulate matter less than 10 microns (PM10) 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 PM10 increases vulnerability to COVID-19 disease. Objective To assess the effect of traffic-derived PM10 on human airway epithelial cell ACE2 expression in vitro. Methods PM10 was collected from Marylebone Road (London) using a kerbside impactor. A549 and human primary nasal epithelial cells were cultured with PM10 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 PM10 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 PM10 concentration (10 μg/mL), found increased ACE2 expression in both A549 cells (control vs. PM10, 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 PM10 increases the expression of the receptor for SARS-CoV-2 in human respiratory epithelial cells. 6 expression may be upregulated by mediators. For example, culture of primary human airway cells with interferon alpha 2 in vitro increases ACE2 transcripts (6) . Since the effect of traffic-related PM on the expression of ACE2 in human airway cell populations is not known we sought, in this study, to assess ACE2 expression in human airway epithelial cells exposed to traffic-derived PM 10 in vitro. Traffic-derived PM 10 was collected as dry particles using a high-volume cyclone placed within 2 metres of Marylebone Road, London, UK (8). Marylebone Road is one of the most polluted roads in Europe, with diesel trucks dominating near-road traffic-derived PM 10 emissions (9). In order to obtain milligram amounts of PM 10 , sampling was done between 6 to 8 h per day on 10 occasions between May and September 2019 (i.e. before the UK lockdown). PM 10 samples were pooled and stored at room temperature in a sterile glass container. An aliquot of PM 10 was diluted in Dulbeccos phosphate-buffered saline (DPBS) to a final concentration of 1 mg/mL and stored as a master stock at -20°C. Cigarette smoke extract (CSE) was collected onto a cotton filter through a peristaltic pump (Jencons Scientific Ltd., East Grinstead, UK) at a fixed rate from two Malborough red cigarettes, as previously described (10) . Cigarette smoke extract was extracted after vortexing in 2 mL Dulbecco's DPBS and stored at -80 0 C as 100% master stock. The human alveolar type II epithelial cell line A549 was purchased from Sigma- Culture of A549 cells with fossil-fuel derived PM 10 (0 to 20 µg/mL) for 2 h resulted in a concentration-dependent increase in ACE2 expression, with significant increase at both 10 µg/mL and 20 µg/mL (n=5, P<0.05, P<0.01 vs. medium control, Figure 1 ). At 20 µg/mL ACE2 increased by 16150 fold (IQR 2577 to 64758). Using a single concentration of PM 10 of 10 µg/mL, ACE2 expression increased in Figure 2B ). Culture of A549 cells with 5% CSE, a putative positive control, increased ACE2 expression (MFI, n=4, 0 (0 to 28) vs. 9088 (7557 to 15831), P<0.05, Figure 3 ). In this study we found that PM 10 , collected next to a major London road dominated by diesel traffic (8), upregulates ACE2 expression in a human type II pneumocyte cell line (A549 cells). We also found that traffic-derived PM 10 upregulates ACE2 expression in human primary nasal epithelial cells, suggesting that this response occurs throughout the respiratory tract. One strength of the present study is that collection of traffic-derived PM 10 by a high-volume cyclone obviated the need to extract PM from filters in solution, and we could therefore accurately determine PM 10 concentrations used in cell culture studies. Although the effect of PM 10 on ACE2 expression in human airway cells has not previously been reported, our findings are compatible with an animal study that reported lung ACE2 protein expression in wild type mice increased by 1.3 fold at 2 days post intratracheal instillation of urban PM 2.5 (11) . A putative protective effect of increased pulmonary ACE2 was suggested in this mouse model by complete recovery of PM-induced acute lung injury in wild type mice, and incomplete recovery in ACE2 knockout mice (11) . We therefore speculate that increased ACE2 expression may, on one hand, be a beneficial response to PM exposure, but on the other hand presents a Trojan horse to the SARS-CoV-2 virus. We included CSE as a putative positive control, since Leung et al (12) There are limitations to this study. First, we did not determine whether increased In conclusion, this study provides the first mechanistic evidence that traffic-derived air pollution increases ACE2 expression in human airway cells and therefore 1 3 vulnerability to SARS-CoV-2 infection. We conclude that there is biological plausibility for epidemiological studies reporting an association between either PM 10 or active smoking and COVID-19 disease. 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