Figure 6.

In vivo administration of ambroxol prevents infection of nasal epithelial cells with pp-VSV-SARS-CoV-2 spike ex vivo. Nasal epithelial cells were obtained from volunteers. The volunteers then inhaled 2 ml ambroxol, that is, 15 mg ambroxol. Nasal epithelial cells were obtained from the opposite nasal cavity 1 h after inhalation. All cells were immediately washed after isolation, and the cell pellets were resuspended in cell culture medium and infected with pp-VSV-SARS-CoV-2 spike or left uninfected for 30 min (A–C) to determine acid sphingomyelinase (A), ceramide (B), and ceramide-enriched membrane domains with ACE2 clusters (C) or for 1 h (D) to determine infection (D). A, to determine acid sphingomyelinase activity, cell pellets were lysed in 50 mM sodium acetate (pH 5.0) and 0.2% NP-40, and the activity of the acid sphingomyelinase was determined by measuring the consumption of added [¹⁴C]sphingomyelin. B, cells were organically extracted, and ceramide was determined by the ceramide kinase assay method. Black symbols indicate C16/C18 ceramides, and gray symbols indicate C22/C24 ceramides. C, freshly isolated nasal epithelial cells were infected with pp-VSV-SARS-CoV-2 for 30 min, washed, fixed in 1% PFA for 10 min, washed and stained with Cy3-coupled anti-ceramide and FITC–anti-ACE2 antibodies. Shown are representative results from four independent experiments. D, cells were infected with pp-VSV-SARS-CoV-2 spike for 1 h, washed, and expression of eGFP was determined after 24 h in at least 500 epithelial cells per sample in randomly chosen microscopic fields. If indicated, C16 ceramide (10 μM) was reconstituted during the infection. A, B, and D show the means ± SD from four volunteers. ∗∗∗p < 0.001, ANOVA, followed by post hoc Student's t tests. ACE2, angiotensin-converting enzyme 2; Amb, ambroxol inhalation; eGFP, enhanced GFP; NP-40, Nonidet P40; PFA, paraformaldehyde; pp-VSV-SARS-CoV-2.