Curbside particulate matter and susceptibility to SARS-CoV-2 infection

Abstract

Background: Biologic plausibility for the association between exposure to particulate matter (PM) less than 10 μm in aerodynamic diameter (PM₁₀) and coronavirus disease 2019 (COVID-19) morbidity in epidemiologic studies has not been determined. The upregulation of angiotensin-converting enzyme 2 (ACE2), the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) entry receptor on host cells, by PM₁₀ is a putative mechanism.

Objective: We sought to assess the effect of PM₁₀ on SARS-CoV-2 infection of cells in vitro.

Methods: PM₁₀ from the curbside of London's Marylebone Road and from exhaust emissions was collected by cyclone. A549 cells, human primary nasal epithelial cells (HPNEpCs), SARS-CoV-2-susceptible Vero-E6 and Calu3 cells were cultured with PM₁₀. ACE2 expression (as determined by median fluorescent intensity) was assessed by flow cytometry, and ACE2 mRNA transcript level was assessed by PCR. The role of oxidative stress was determined by N-acetyl cysteine. The cytopathic effect of SARS-CoV-2 (percentage of infection enhancement) and expression of SARS-CoV-2 genes' open reading frame (ORF) 1ab, S protein, and N protein (focus-forming units/mL) were assessed in Vero-E6 cells. Data were analyzed by either the Mann-Whitney U test or Kruskal-Wallis test with the Dunn multiple comparisons test.

Results: Curbside PM₁₀ at concentrations of 10 μg/mL or more increased ACE2 expression in A549 cells (P = .0021). Both diesel PM₁₀ and curbside PM₁₀ in a concentration of 10 μg/mL increased ACE2 expression in HPNEpCs (P = .0022 and P = .0072, respectively). ACE2 expression simulated by curbside PM₁₀ was attenuated by N-acetyl cysteine in HPNEpCs (P = .0464). Curbside PM₁₀ increased ACE2 expression in Calu3 cells (P = .0256). In Vero-E6 cells, curbside PM₁₀ increased ACE2 expression (P = .0079), ACE2 transcript level (P = .0079), SARS-CoV-2 cytopathic effect (P = .0002), and expression of the SARS-CoV-2 genes' ORF1ab, S protein, and N protein (P = .0079).

Conclusions: Curbside PM₁₀ increases susceptibility to SARS-COV-2 infection in vitro.

Keywords: PM10; Particulate matter; SARS-CoV-2; airway epithelial cells.

Fig 1

Effect of PM₁₀ on ACE2 expression. PM₁₀ was collected from the curbside of Marylebone Road (curbside PM₁₀) and the exhaust of a diesel car (diesel PM₁₀) by using the same cyclone. Cells were cultured with PM₁₀ for 2 hours, and ACE2 expression was determined by flow cytometry. The results are expressed as mean fluorescence intensity (MFI) adjusted for isotypic antibody control. A, Dose-response relationship between curbside PM₁₀ and ACE2 expression by A549 cells. Two data points are omitted for visual convenience. B, Replication of A549 dose-response data using 10 μg/mL of curbside PM₁₀. C, Effect of curbside PM₁₀ and diesel PM₁₀ (10 μg/mL) on ACE2 expression (MFI) in purchased (Promocell) HPNEpCs. D, Effect of curbside PM₁₀ (10 μg/mL) on ACE2 expression in primary nasal epithelial cells obtained from a local adult donor HPNEpCs. E, Effect of curbside PM₁₀ (10 μg/mL) on ACE2 expression (MFI) in Calu3 cells. F, Effect of the antioxidant N-acetyl cysteine on expression of curbside PM₁₀-stimulated ACE2 expression (MFI) in purchased HPNEpCs. Columns represent medians from at least 4 separate experiments. Data were compared by either the Mann-Whitney U test or Kruskal-Wallis test and Dunn multiple comparisons test.

Fig 2

Effect of 5% CSE on ACE2 expression in A549 cells (A) and purchased (from Promocell) HPNEpCs (B). Results are expressed as mean fluorescence intensity (MFI) adjusted for isotypic antibody control. Columns represent medians from at least 4 separate experiments, and data are compared by the Mann-Whitney U test.

Fig 3

Effect of curbside PM₁₀ (10 μg/mL) for 2 hours on Vero-E6 cell. A, ACE2 expression by flow cytometry expressed as mean fluorescence intensity (MFI) adjusted for isotypic antibody control. B, ACE2 transcript level. Threshold cycle values were normalized to glyceraldehyde 3-phosphate dehydrogenase, and relative quantification was analyzed by using the 2^-ΔΔCt method. Columns represent medians from 5 separate experiments, and data are compared by the Mann-Whitney U test.

Fig 4

Effect of curbside PM₁₀ on SARS-CoV-2 infection of Vero-E6 cells. A, SARS-CoV-2 CPE expressed as infection enhancement (percentage) compared with the control medium. Data from 7 separate experiments are compared by the Kruskal-Wallis test and Dunn multiple comparisons test. Effect of curbside PM₁₀ on SARS-CoV-2 viral gene expression in Vero-E6 cells: ORF 1ab (B), S protein (C), and N protein (D). Viral protein data from 5 separate experiments are expressed as FFU/mL calculated from a standard curve with an input of 1.5 × 10⁶ FFU/mL and serial 10-fold dilutions of SARS-CoV-2 viral stock. Ct values are normalized to the standard curve for quantification. Columns represent medians from 5 separate experiments, and data are compared by using the Mann-Whitney U test.