Cigarette Smoke Stimulates SARS-CoV-2 Internalization by Activating AhR and Increasing ACE2 Expression in Human Gingival Epithelial Cells

Abstract

A large body of evidence shows the harmful effects of cigarette smoke to oral and systemic health. More recently, a link between smoking and susceptibility to coronavirus disease 2019 (COVID-19) was proposed. COVID-19 is due to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which uses the receptor ACE2 and the protease TMPRSS2 for entry into host cells, thereby infecting cells of the respiratory tract and the oral cavity. Here, we examined the effects of cigarette smoke on the expression of SARS-CoV-2 receptors and infection in human gingival epithelial cells (GECs). We found that cigarette smoke condensates (CSC) upregulated ACE2 and TMPRSS2 expression in GECs, and that CSC activated aryl hydrocarbon receptor (AhR) signaling in the oral cells. ACE2 was known to mediate SARS-CoV-2 internalization, and we demonstrate that CSC treatment potentiated the internalization of SARS-CoV-2 pseudovirus in GECs in an AhR-dependent manner. AhR depletion using small interference RNA decreased SARS-CoV-2 pseudovirus internalization in CSC-treated GECs compared with control GECs. Our study reveals that cigarette smoke upregulates SARS-CoV-2 receptor expression and infection in oral cells. Understanding the mechanisms involved in SARS-CoV-2 infection in cells of the oral cavity may suggest therapeutic interventions for preventing viral infection and transmission.

Keywords: ACE2; AhR; COVID-19; SARS-CoV-2; TMPRSS2; cigarette smoke; smoking.

Figure 1

Cigarette smoke condensate treatment increases angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) levels in human gingival epithelial cells. Human gingival epithelial cells (GECs) were treated with or without cigarette smoke condensates (CSC) at different concentrations for 24 h, and expression of ACE2 and TMPRSS2 were determined by Western blot. (A) shows a representative Western blot image for ACE2 and TMPRSS2 in untreated GECs. (B) shows representative Western blot images for ACE2 and (D) TMPRSS2 after treatment with CSC at different concentrations for 24 h. (C,E) Graphs show average ± SEM of band intensity (arbitrary units) for ACE2 (C) and TMPRSS2 (E). (A) n = 3, (B,C) n = 7, (D,E) n = 6. * p ≤ 0.05; ** p ≤ 0.01.

Figure 2

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment increases angiotensin-converting enzyme 2 (ACE2) levels, but not transmembrane serine protease 2 (TMPRSS2), in human gingival epithelial cells. GECs were treated with or without cigarette smoke condensates (CSC) at different concentrations as indicated, or with TCDD (10 nM), for 24 h. Expression of ACE2 and TMPRSS2 were determined by Western blot. (A) shows representative Western blots for ACE2 and TMPRSS2 in GECs after treatment with or without CSC or TCDD. (B,C) Graphs show average ± SEM of band intensity (arbitrary units) for ACE2 (B) and TMPRSS2 (C). Data shown are representative of at least 3 independent experiments. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Figure 3

Aryl hydrocarbon receptor (AhR) is activated by treatment with cigarette smoke condensate in gingival epithelial cells. Human gingival epithelial cells (GECs) were treated with or without cigarette smoke condensates (CSC) at different concentrations as indicated, or with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 10 nM), for 24 h. AhR expression and activation was determined by Western blot and immunofluorescence microscopy, respectively. (A) shows a representative Western blot for AhR in unstimulated GECs. (B) shows representative images of immunofluorescence for AhR. (C) Graph shows average percentage ± SEM of AhR translocation to the nucleus by measuring the intensity of fluorescence using Image J. (A–C) n = 3. * p ≤ 0.05.

Figure 4

Pretreatment with cigarette smoke condensates increases severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus infection in gingival epithelial cells. Human gingival epithelial cells (GECs) were treated with or without cigarette smoke condensates at different concentrations as indicated, or with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 10 nM), for 24 h, prior to infection with SARS-CoV-2 pseudotyped GFP-tagged lentivirus for an additional 24 h. SARS-CoV-2 pseudovirus infection was visualized by immunofluorescence microscopy. (A) shows representative images of immunofluorescence for GFP-tagged SARS-CoV-2 pseudovirus. (B) Graph shows average percentage ± SEM of GFP fluorescence intensity, measured using Image J. (A,B) n = 3. * p ≤ 0.05; *** p ≤ 0.001. Diameter bar = 100 μm.

Figure 5

Treatment with cigarette smoke condensates increases severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus infection in gingival epithelial cells via aryl hydrocarbon receptor (AhR) signaling. Human gingival epithelial cells (GECs) were transfected with siRNA sequences against AhR or an unrelated target, and treated with or without cigarette smoke condensate (10 μg/mL) for 24 h, prior to infection with SARS-CoV-2 pseudotyped GFP-tagged lentivirus for an additional 24 h. SARS-CoV-2 pseudotyped GFP-tagged lentivirus infection was visualized by immunofluorescence microscopy. (A) shows representative images of immunofluorescence for GFP-tagged SARS-CoV-2 pseudovirus. (B) Graph shows average percentage ± SEM of GFP fluorescence intensity, measured using Image J. (A,B) n = 3. * p ≤ 0.05; ** p ≤ 0.01; Diameter bar = 100 μm.

Figure 6

Proposed scheme for the mechanism involved in cigarette smoke-induced enhancement of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in human gingival epithelial cells. Cigarette smoke activates aryl hydrocarbon receptor (AhR) in human gingival epithelial cells, which increases the surface levels of angiotensin-converting enzyme 2 (ACE2). Increased expression of ACE2 facilitates SARS-CoV-2 infection of the oral epithelial cells.