Thermal Water Reduces the Inflammatory Process Induced by the SARS-CoV-2 Spike Protein in Human Airway Epithelial Cells In Vitro

Abstract

Background: Although treatments using thermal water have yielded beneficial effects in respiratory tract infections, the effects of thermal water under experimental conditions similar to those triggered by SARS-CoV-2 have yet to be evaluated. This study aimed to assess whether thermal water could interfere with the interaction between SARS-CoV-2 and host cells and influence inflammatory factors. Methods: Human nasal epithelial primary cells (HNEpCs) were stimulated with SARS-CoV-2 spike protein in the presence or absence of thermal water or tap water. Cell viability, cytokine concentration, ACE2 and TMPRSS2 levels, and ACE2 activity were determined in the cell cultures. Results: Exposure of HNEpCs to spike protein increased IL-6, IL-8, and IL-1β production, with decreased production observed in the presence of thermal water at an optimal dose. Treatment of cells with tap water did not affect cytokine release in unstimulated or spike-stimulated cells. Spike-protein-stimulated HNEpCs showed reduced levels of ACE2, which were partially restored only in the presence of thermal water. Spike protein did not affect the TMPRSS2 levels of the cell lysates. Stimulation with spike protein induced an increase in the concentration of both receptors in the supernatants, while treatment with thermal water reduced TMPRSS2 levels in both the cells and supernatants. Stimulation with spike protein increased ACE2 activity, which was reduced with thermal water. Conclusions: This study shows the regulatory effects of mineral-rich thermal water on spike-protein-induced pro-inflammatory cytokine production and the amount and activity of receptors mainly involved in viral entry, suggesting a potential use of this treatment as a support therapy for SARS-CoV-2 infection of the upper respiratory tract.

Keywords: COVID-19; SARS-CoV-2; balneotherapy; inflammation; spike protein; thermal water.

Figure 1

Effect of thermal water on cytokine production in human nasal epithelial primary cells (HNEpCs). The cells were treated with increasing percentages of thermal water (H₂O). Cell viability was then evaluated after 24 h (a). Cells were stimulated (or not) for 24 h with 1 µg/mL SARS-CoV-2 spike protein in the presence or absence of 1% thermal water or tap water. Culture supernatants were analyzed for the production of IL-6 (b), IL-8 (c), and IL-1β (d). Results are presented as the mean ± SD of four separate experiments. * p < 0.05 vs. untreated (UNT) cells; # p < 0.05 vs. spike-protein-stimulated cells; § p < 0.05 vs. spike-protein-stimulated cells treated with thermal water.

Figure 2

Effect of thermal water on the levels of SARS-CoV-2 spike protein receptors. Human nasal epithelial primary cells (HNEpCs) were stimulated (or not stimulated) for 24 h with 1 µg/mL SARS-CoV-2 spike protein in the presence or absence of 1% thermal water (H₂O) or tap water. The concentrations of ACE2 and TMPRSS2 were determined in cell lysates (a,b). In addition, ACE2 and TMPRSS2 levels were determined in culture supernatants (c,d). Results are presented as the mean ± SD of four separate experiments. * p < 0.05 vs. untreated (UNT) cells; # p < 0.05 vs. spike-protein-stimulated cells; § p < 0.05 vs. spike-protein-stimulated cells treated with thermal water.

Figure 3

Effect of thermal water on ACE2 activity in human nasal epithelial primary cell (HNEpC) cultures. HNEpCs were stimulated (or not) with 1 µg/mL SARS-CoV-2 spike protein in the presence or absence of 1% thermal water (H₂O) or tap water. ACE2 activity was evaluated after 24 h in cells (a) and supernatants (b). Results are presented as the mean ± SD of four separate experiments. * p < 0.05 vs. untreated (UNT) cells; # p < 0.05 vs. spike-protein-stimulated cells; § p < 0.05 vs. spike-protein-stimulated cells treated with thermal water.