Modulator Combination Improves In Vitro the Microrheological Properties of the Airway Surface Liquid of Cystic Fibrosis Airway Epithelia

Abstract

Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a plasma membrane protein expressed on the apical surface of secretory epithelia of the airways. In the airways, defective or absent function of the CFTR protein determines abnormalities of chloride and bicarbonate secretion and, in general, of the transepithelial homeostasis that lead to alterations of airway surface liquid (ASL) composition and properties. The reduction of ASL volume impairs ciliary beating with the consequent accumulation of a sticky mucus. This situation prevents normal mucociliary clearance, favoring the survival and proliferation of bacteria and contributing to the genesis of the CF pulmonary disease. We explored the potential of some CFTR modulators, namely ivacaftor, tezacaftor, elexacaftor and their combination Kaftrio^TM, capable of partially recovering the basic defects of the CFTR protein, to ameliorate the transepithelial fluid transport and the viscoelastic properties of the mucus when used singly or in combination. Primary human bronchial epithelial cells obtained from CF and non-CF patients were differentiated into a mucociliated epithelia in order to assess the effects of correctors tezacaftor, elexacaftor and their combination with potentiator ivacaftor on the key properties of ASL, such as fluid reabsorption, viscosity, protein content and pH. The treatment of airway epithelia bearing the deletion of a phenylalanine at position 508 (F508del) in the CFTR gene with tezacaftor and elexacaftor significantly improved the pericilial fluid composition, reducing the fluid reabsorption, correcting the ASL pH and reducing the viscosity of the mucus. Kaftrio^TM was more effective than single modulators in improving all the evaluated parameters, demonstrating once more that this combination recently approved for patients 6 years and older with cystic fibrosis who have at least one F508del mutation in the CFTR gene represents a valuable tool to defeat CF.

Keywords: bronchial epithelial cell culture; correctors; cystic fibrosis; ion transport; pericilial mucus properties.

Figure 1

(a) Transepithelial conductance, G, in HBEC epithelial monolayers expressing WT (non-CF) and F508del mutant CFTR (CF). Measurements (n = 10 for both conditions) were obtained after treatment with DMSO as vehicle. (b) Difference between transepithelial conductances measured before and after the different treatments (∆G), measured in epithelia expressing the F508del mutation. (DMSO: 0.1% DMSO (vehicle control), iva: 1 µM of ivacaftor, tez: 5 µM of tezacaftor, ele: 5 µM of elexacaftor, mix: 1 µM of ivacaftor + 5 µM of tezacaftor + 5 µM of elexacaftor). The sample size, n, was comprised between 6 ≤ n ≤ 9. Asterisks indicate that the ∆G value is significantly different from that measured in CF HBEC treated with the DMSO vehicle.

Figure 2

Fluid reabsorption, J_W, of HBEC monolayers, cultured on Transwell permeable supports, from non-CF cells treated with 0.1% DMSO (vehicle control) and CF HBEC treated with 0.1% DMSO (vehicle control), 1 µM of ivacaftor (iva), 5 µM of tezacaftor (tez), 5 µM of elexacaftor (ele), 1 µM of ivacaftor + 5 µM of tezacaftor + 5 µM of elexacaftor (Mix) for 48 h. Fluid reabsorption of each sample was evaluated gravimetrically as described in the Section 4. The white circles represent the values of each individual measure (6 ≤ n ≤ 12). The section mark (§) indicates data that are statistically different from non-CF control cells, while asterisks (*) indicate a statistical significance versus control CF cells.

Figure 3

Micro-rheology of the airway surface liquid (ASL) recovered from HBEC monolayers measured by the multiple particle technique (MPT). (a) Viscosity of the ASL collected from the apical side of non-CF and CF epithelia. Non-CF HBEC epithelia were incubated with a solution containing 0.1% DMSO (vehicle control) while CF HBEC epithelia were treated with 0.1% DMSO (vehicle control), 1 µM of ivacaftor (iva), 5 µM of tezacaftor (tez), 5 µM of elexacaftor (ele), 1 µM of ivacaftor + 5 µM of tezacaftor + 5 µM of elexacaftor (mix), respectively, administered on the apical side of the monolayers. The white circles in (a) represent the values of each single measure (10 ≤ n ≤ 12). The section mark (§) indicates that data are statistically different from non-CF control cells, while asterisks (*) indicate a statistical significance versus control CF cells. (b–g) Plots of the square displacement, <msd> against the time interval of ≤100 beads in the ASL samples from non-CF HBEC (WT) treated with DMSO (b) and from F508del mutant CF HBEC (F508del) treated with DMSO (c), ivacaftor (VX770) (d), tezacaftor (VX661) (e), elexacaftor (VX445) (f), and ivacaftor + tezacaftor + elexacaftor (VX661 + VX445 + VX770) (g). The average <msd> is shown as a black solid line. The values of viscosity, η, the elastic modulus, α, and the sample size, n, are indicated in each panel. The insets in panels (b–g) show the trajectory path of a bead recorded in each ASL sample.

Figure 4

ASL pH measured from non-CF HBEC epithelia treated with 0.1% DMSO (vehicle control) and from CF HBEC epithelia incubated with 0.1% DMSO (vehicle control), 1 µM of ivacaftor (iva), 5 µM of tezacaftor (tez), 5 µM of elexacaftor (ele), and 1 µM of ivacaftor + 5 µM of tezacaftor + 5 µM of elexacaftor (mix), respectively. The white circles represent the values of each single measure. The section mark (§) indicates that data are statistically different from non-CF control cells, while asterisks (*) indicate a statistical significance compared to control CF cells.