Betamethasone prevents human rhinovirus- and cigarette smoke- induced loss of respiratory epithelial barrier function

Abstract

The respiratory epithelium is a barrier against pathogens and allergens and a target for therapy in respiratory allergy, asthma and chronic obstructive pulmonary disease (COPD). We investigated barrier-damaging factors and protective factors by real-time measurement of respiratory cell barrier integrity. Barrier integrity to cigarette smoke extract (CSE), house dust mite (HDM) extract, interferon-γ (IFN-γ) or human rhinovirus (HRV) infection alone or in combination was assessed. Corticosteroids, lipopolysaccharide (LPS), and nasal mucus proteins were tested for their ability to prevent loss of barrier integrity. Real-time impedance-based measurement revealed different patterns of CSE-, HDM-, IFN-γ- and HRV-induced damage. When per se non-damaging concentrations of harmful factors were combined, a synergetic effect was observed only for CSE and HDM. Betamethasone prevented the damaging effect of HRV and CSE, but not damage caused by HDM or IFN-γ. Real-time impedance-based measurement of respiratory epithelial barrier function is useful to study factors, which are harmful or protective. The identification of a synergetic damaging effect of CSE and HDM as well as the finding that Betamethasone protects against HRV- and CSE-induced damage may be important for asthma and COPD.

Figure 1

Comparison of two different methods to determine the influence of HDM extract, interferon-γ (IFN-γ), cigarette smoke extract (CSE) and human rhinovirus 14 (HRV 14) infection on barrier function in primary nasal epithelial cells. Primary human nasal epithelial cells were grown in a 2-chamber tissue culture model (A–D, top panels) or on E-plates in the xCELLigence system (A–D, bottom panels). Changes of trans-epithelial resistance (TER, y-axes, top panels) or of cell impedance (normalised Cell Index, y-axes, bottom panels) were determined at indicated time points (x-axis) after exposure of cells to different concentrations of HDM extract (A) IFN-γ (B) CSE (C) or HRV 14 (D). Results from three independent experiments performed in duplicates are shown. Statistical significance analyses were performed with the two-sided Welch-tests. Standard errors of the mean values are displayed as error bars for each measuring point.

Figure 2

Effect of four different damaging factors on primary human nasal epithelial cells and on the respiratory epithelial cell line 16HBE14o-. Primary cells (top panels) and the respiratory cell line (bottom panels) were exposed to different concentrations of HDM (A) interferon-γ (B) standardised cigarette smoke extract (C) or human rhinovirus 14 (D). Data were normalised at the time of addition of factors (time point 0). Impedance values were measured every 30 minutes by the xCELLigence system for 48 hours (A,C,D) or 240 hours (B) (x-axes) and are expressed as a normalised Cell Index (y-axes). Results are derived from three independent experiments which were done in duplicates. Statistical significance analyses were performed with the two-sided Welch-tests. Standard errors of the mean values are displayed as error bars for each time point in (A,C,D) while in (B) the standard error of the mean is shown only for every 5^th measuring point for better visualisation.

Figure 3

Synergetic effect of non-damaging concentrations of cigarette smoke and HDM extract on barrier function. 16HBE14o- cells grown in the xCELLigence system were exposed to either 12.5 µg/mL of HDM extract, or 1% of cigarette smoke extract, or a combination, or a medium control (untreated). Data were normalised at time point 0 and impedance values (y-axis: normalised Cell Index) were then measured every 30 minutes by the xCELLigence system for 48 hours (x-axis). Results are derived from three independent experiments performed in duplicates. Statistical significance analyses were performed with the two-sided Welch-tests. Standard errors of the mean values are displayed as error bars for each measuring point.

Figure 4

Betamethasone prevents cigarette smoke- and rhinovirus-induced damage of respiratory epithelial cells. 16HBE14o- cells were incubated at time point 0 with Betamethasone (3 µg/mL) or medium. Three hours thereafter, either 4% cigarette smoke extract (A) was added to the cells or cells were infected with 150 TCID₅₀/cell of rhinovirus 14 (B). Data were normalised at time point 0 and impedance values (y-axis: normalised Cell Index) were measured every 30 minutes for 48 hours. Results from three independent experiments performed in duplicates are shown. Statistical significance analyses were performed with the two-sided Welch-tests. Standard errors of the mean values are visualised as error bars.