Novel Therapy of Bicarbonate, Glutathione, and Ascorbic Acid Improves Cystic Fibrosis Mucus Transport

Abstract

Defective airway mucus clearance is a defining characteristic of cystic fibrosis lung disease, and improvements to current mucolytic strategies are needed. Novel approaches targeting a range of contributing mechanisms are in various stages of preclinical and clinical development. ARINA-1 is a new nebulized product comprised of ascorbic acid, glutathione, and bicarbonate. Using microoptical coherence tomography, we tested the effect of ARINA-1 on central features of mucociliary clearance in F508del/F508del primary human bronchial epithelial cells to assess its potential as a mucoactive therapy in cystic fibrosis. We found that ARINA-1 significantly augmented mucociliary transport rates, both alone and with CFTR (cystic fibrosis transmembrane conductance regulator) modulator therapy, whereas airway hydration and ciliary beating were largely unchanged compared with PBS vehicle control. Analysis of mucus reflectivity and particle-tracking microrheology indicated that ARINA-1 restores mucus clearance by principally reducing mucus layer viscosity. The combination of bicarbonate and glutathione elicited increases in mucociliary transport rate comparable to those seen with ARINA-1, indicating the importance of this interaction to the impact of ARINA-1 on mucus transport; this effect was not recapitulated with bicarbonate alone or bicarbonate combined with ascorbic acid. Assessment of CFTR chloride transport revealed an increase in CFTR-mediated chloride secretion in response to ARINA-1 in CFBE41o^- cells expressing wild-type CFTR, driven by CFTR activity stimulation by ascorbate. This response was absent in CFBE41o^- F508del cells treated with VX-809 and primary human bronchial epithelial cells, implicating CFTR-independent mechanisms for the effect of ARINA-1 on cystic fibrosis mucus. Together, these studies indicate that ARINA-1 is a novel potential therapy for the treatment of impaired mucus clearance in cystic fibrosis.

Keywords: bicarbonate; cystic fibrosis; glutathione; mucus transport; mucus viscosity.

Figure 1.

ARINA-1 improves mucus transport in F508del-homozygous primary human bronchial epithelial (HBE) cells. (A–H) Representative images of F508del/F508del HBE monolayers treated with PBS at 0 hours pretreatment (A and B), PBS at 6 hours (C and D), ARINA-1 at 0 hours pretreatment (E and F), and ARINA-1 at 6 hours (G and H). (A, C, E, and G) Text designates the epithelial cell monolayer (ep), mucus layer (mu), air interface (air), and cross-sectional view of the filter membrane (Filter). The yellow bar indicates airway surface liquid (ASL) layer depth, and the blue arrows denote cilia. Scale bars, 20 μm. (B, D, F, and H) Resliced views of images in which mucociliary transport (MCT) rate was measured as viewed from the apical surface. Orientation of the images is altered such that the slope of the diagonal streak (represented by the red arrow) represents the vectoral transport of mucus particles over time (time = y-axis). This enables visualization of differences in MCT rate (measured in video rate) using still images: steeper slope = slower transport; more horizontal slope = faster transport. (I–K) Change from baseline versus PBS in MCT rate (I), ASL depth (J), and ciliary beat frequency (CBF) (K) after 6 and 24 hours of treatment with PBS or ARINA-1; n = 44–45 monolayers per condition across four different donors with cystic fibrosis (CF). (L) Change from baseline versus PBS in MCT rate upon cotreatment with lumacaftor and ivacaftor. Two-way ANOVA and Tukey’s post hoc test. n = 20 monolayers per condition across three different donors with CF. Mean ± SEM. ****P < 0.0001 versus PBS control.

Figure 2.

ARINA-1 mucus clearance effect exceeds equiosmolar treatments with hypertonic saline. Equiosmolar treatments of F508del/F508del HBE monolayers with normosmolar saline/normosmolar ARINA-1, medium osmolar saline/medium osmolar ARINA-1, and high osmolar saline/high osmolar ARINA-1 were performed as in Table 1. (A–F) Representative reslices of airway mucus transport (as in Figure 1) for each treatment condition at 6 hours after treatment. Slope of the diagonal streak (represented by the red arrow) represents the vectoral transport of mucus particles over time (time = y-axis [see Figures 1B, 1D, 1F, and 1H]), enabling visualization of differences in MCT rate. (G) Change in ASL depth from baseline at 6 and 24 hours after treatment. Top asterisks: high osmolar ARINA 1 versus normosmolar saline at same time point; bottom asterisks: high osmolar saline versus normosmolar saline at same time point, by two-way ANOVA and Tukey’s post hoc test. *P < 0.05, **P < 0.01, and ****P < 0.0001. (H) Paired comparisons of change in ASL depth at 6 hours of equiosmolar conditions (each point represents mean change per monolayer). ns = not significant. (I) Change in MCT rate relative to normosmolar saline and compared with baseline. (J) Change in MCT rate between dual groups of equiosmolar conditions at 6 hours, each point representing mean change from baseline per monolayer. One-way ANOVA and Tukey’s post hoc test. n = 7–8 monolayers per condition across two different donors with CF. Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 3.

Changes in ASL depth and ciliary beating with ARINA-1 predict changes in mucus transport. (A) Linear regression analysis of the change in ASL depth versus the change in MCT at 6 hours for ARINA-1 and PBS-treated F508del/F508del HBE monolayers with (B) comparison of regression slopes. (C) Linear regression analysis of change in CBF versus change in MCT at 6 hours for ARINA-1– and PBS-treated monolayers with (D) comparison of regression slopes by unpaired two-tailed t test. n = 44–45 monolayers per condition across four different donors with CF. The ARINA-1 linear slopes are compatible with a normal relationship between ASL depth and CBF with MCT, often absent in CF. Mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4.

ARINA-1 decreases reflectance intensity. Representative attenuation-corrected images of F508del/F508del HBE monolayers treated with (A) PBS or (B) ARINA-1 for 6 hours. Text designates the epithelial cell monolayer (ep), air interface (air), and cross-sectional view of the filter membrane (Filter). The yellow box circumscribes the ASL layer selected for analysis, and the blue arrows denote representative cilia. Scale bars, 20 μm. (C) Reflectivity histogram of the attenuation-corrected image plotted by treatment condition. Leftward shift of the histogram indicates a lower proportion of highly intense pixels, which has been shown to be proportionate to mucus viscosity. (D) Mean intensity of airway mucus plotted by treatment condition. **P < 0.01 and ***P < 0.001.

Figure 5.

ARINA-1 decreases effective viscosity. (A) Representative tracings of beads in the mucus layer of F508del/F508del HBE monolayers treated with PBS or ARINA-1 for 6 hours as assessed by particle-tracking microrheology. (B) Plot of mean squared displacement (MSD) of each bead over time with (C) corresponding viscoelastic curves at frequency sweep ranging from 0.46 to 17.36 Hz. Statistics reported for comparison of ARINA-1 versus PBS. Two-way ANOVA and Sidak’s multiple comparisons test. n = 3–5 monolayers per condition from one donor with CF. Note logarithmic scale x- and y-axes. Mean ± SEM. ****P < 0.001. (D) Relationship between effective viscosity (at 1 Hz) and reflectance intensity. Data for same cells as in Figure 4 with four or five reflectance intensity replicates per well. Semilogarithmic regression relationship (dotted line) with R² = 0.44; P = 0.10; n = 7. Note logarithmic scale x-axis.

Figure 6.

The combination of bicarbonate with glutathione (GSH) recapitulates the effect of ARINA-1 on mucus transport. F508del/F508del HBE monolayers were treated with PBS vehicle control, ARINA-1, bicarbonate, bicarbonate + GSH, or bicarbonate + ascorbate (ASC) for 6 or 24 hours. (A) Change from baseline versus PBS in MCT rate by two-way ANOVA and Tukey’s post hoc test. At 6 hours, top asterisks = ARINA-1; bottom asterisks = bicarbonate + GSH (color-coded). (B) Change in MCT rate from baseline at 6 hours by treatment condition, with each point representing mean change per monolayer. One-way ANOVA and Tukey’s post hoc test. Change from baseline versus PBS in (C) ASL depth and (D) CBF. (E) Linear regression analysis of change in ASL depth versus change in MCT at 6 hours across treatment conditions with (F) comparison of regression slopes (one-way ANOVA = not significant; Bartlett’s test of homoscedasticity, P < 0.0001). (G) Linear regression analysis of change in CBF versus change in MCT at 6 hours with (H) comparison of regression slopes by unpaired two-tailed t test. n = 20–21 monolayers per condition across four different donors with CF. Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 7.

ARINA-1 increases CFTR-mediated chloride secretion in wild-type (WT) CFBE41o⁻ cells, but not in F508del-CFTR CFBE41o⁻, WT HBE, or F508del-F508del HBE cells. Transepithelial chloride currents across the epithelia of CFBE41o⁻ cells and HBE monolayers were assessed by Ussing chamber analysis upon treatment with PBS, ARINA-1, or ASC. (A) Representative tracings with (B) summary bar graphs of chloride currents in CFBE41o⁻ cells expressing WT-CFTR. (C) Representative tracings with (D) summary bar graphs of chloride currents in VX-809–corrected F508del-CFTR CFBE41o⁻ cells. (E) Representative tracings with (F) summary bar graphs of chloride currents in WT HBE monolayers. (G) Representative tracings with (H) summary bar graphs of chloride currents in VX-809–corrected F508del-F508del HBE monolayers. One-way ANOVA and Tukey’s post hoc test. CFBE41o⁻ cells: n ≥ 4 monolayers per condition. HBE monolayers: n = 8–12 monolayers per condition across three different donors with CF or WT donors. Mean ± SEM. **P < 0.01, ***P < 0.001, and ****P < 0.0001. Isc = short circuit current. ΔIsc = change in short-circuit current.