Bicarbonate and functional CFTR channel are required for proper mucin secretion and link cystic fibrosis with its mucus phenotype

Abstract

Cystic fibrosis (CF) is caused by a nonfunctional chloride and bicarbonate ion channel (CF transmembrane regulator [CFTR]), but the link to the phenomenon of stagnant mucus is not well understood. Mice lacking functional CFTR (CftrΔ508) have no lung phenotype but show similar ileal problems to humans. We show that the ileal mucosa in CF have a mucus that adhered to the epithelium, was denser, and was less penetrable than that of wild-type mice. The properties of the ileal mucus of CF mice were normalized by secretion into a high concentration sodium bicarbonate buffer (~100 mM). In addition, bicarbonate added to already formed CF mucus almost completely restored the mucus properties. This knowledge may provide novel therapeutic options for CF.

Figure 1.

The ileal mucus of the CftrΔF508 mouse is attached to the epithelium and cannot be easily aspirated. (A) Carnoy-fixed ileal sections from WT (n = 7) and CF (n = 7) mice were immunostained for Muc2 (green) and nuclei (blue). Arrows point to mucus attached to goblet cells. Bars, 100 µm. (B) Transmission electron micrographs of CCh- and PGE₂-stimulated ileal explants of WT (n = 2) and CF (n = 2) mice. Empty denotes space empty from mucus between the epithelial cell microvilli and mucus layer. Note the distended crypt with condensed material in CF mice. Bars, 2 µm. (C) Bright field images of WT mouse ileal tissue mounted in the horizontal chamber with charcoal to visualize the upper surface of the transparent mucus layer before and after aspiration. See Video 1 to watch removal of charcoal labeled mucus (arrow points at easily distinguished crypt opening). (D) Same experiment as in C, but on ileal explant from the CF mouse. See Video 2 to watch the difficulty in removing the thick and opaque mucus covering the villi (arrows). (E) Representative confocal images of a single villus from ileal tissue (red) from WT (n = 3) or CF (n = 4) mice mounted in the horizontal chamber overlaid with 2 µm fluorescent beads (green) for 40 min. Experiment illustrates mucus penetrability. Bars, 50 µm.

Figure 2.

Stimulation of mucus secretion. (A) Mucus thickness of ileal explant mounted in the horizontal chamber. After mounting (time 0), mucus was removed from the CF mice by extensive aspiration before the measurement at 20 min. Mucus secretion was stimulated by basal perfusion with PGE₂ and CCh for 40 min (WT, CF, n = 4; WT CCh-PGE₂, n = 8; CF CCh-PGE₂, n = 7; **, P = 0.008). (B) Total mucus thickness after 40-min stimulation with CCh and PGE₂ and remaining thickness after aspiration of the mucus (n.s., nonsignificant; WT, n = 7; CF, n = 6; ***, P = 0.0006). (C) Quantification of Muc2 amount in secreted mucus from WT and CF ileum. The secreted mucus was collected, analyzed on an SDS-agarose-polyacrylamide composite gel, and visualized by SYPRO Ruby (top) and Alcian blue (bottom). A representative gel of four repeated experiments is shown. The Muc2 monomeric band is indicated by the arrows and has a molecular mass of ∼2.5 MD. (D) Intensity of the SYPRO Ruby staining of the Muc2 monomeric band in CF (n = 6) and WT (n = 6; four gels; **, P = 0.002). Data are presented as mean ± SEM.

Figure 3.

Loss of basolateral HCO₃⁻ transport gives WT a CF mucus phenotype. (A) The mucus from the mounted WT (n = 5) ileal specimens was removed. The apical surface was exposed to buffers with 23 mM NaHCO₃ and the serosal surface exposed to buffers with 23 mM (Control) or 0 mM HCO₃⁻. After 15 min, mucus release was stimulated by serosal exposure to CCh and PGE₂ for 40 min. The total mucus thickness was measured (Total), followed by aspiration of the mucus and measurement of the remaining thickness (Remaining; **, P = 0.008). (B) Bright field images of WT mouse ileal tissue in the absence of serosal HCO₃⁻ before and after mucus aspiration. See Video 3 to watch the difficulty in removing the charcoal-labeled mucus. (C) WT (n = 5) ileal tissue was treated with (5 mM) or without serosal Ba²⁺ before stimulation with CCh and PGE₂. The total mucus thickness was measured (Total), followed by aspiration of the mucus and measurement of the remaining thickness (Remaining; **, P = 0.008). (D) WT ileal tissue was treated with apical or serosal DIDS before stimulation with CCh and PGE₂ (n = 5 in all three groups; *, P < 0.05; **, P < 0.01). Data are presented as mean ± SEM.

Figure 4.

The CF mucus phenotype can be normalized by apical HCO₃⁻. (A) Total CF mucus thickness after CCh- and PGE₂-stimulated secretion into apical buffers containing 23, 69, 92, or 115 mM apical HCO₃⁻. (B) Remaining mucus thickness after aspiration in the presence of increasing concentrations of apical HCO₃⁻ (n = 6 for 23, 92, and apical 115; n = 3 for 69 and 115 serosal; **, P < 0.05). (C) Bright field images of CF ileal tissue with mucus secreted into 115 mM HCO₃⁻ apical buffer before and after aspiration. See Video 4 to watch the aspiration the mucus. (D) Total and remaining mucus thickness after CCh- and PGE₂-stimulated secretion into apical buffers containing 10 or 20 mM EDTA (n = 5 in all three groups; **, P = 0.008). Data are presented as mean ± SEM.

Figure 5.

High concentrations of HCO₃⁻ can normalize already formed mucus. (A) 115 mM, NaHCO₃ was applied apically to mucus present on ileal explants from CF (n = 3) and WT (n = 3) mice. Mucus thickness was measured at indicated time points. (B) Confocal image of the surface of ileal explants from CF mice (n = 4), where the mucus has been exposed to 115 mM NaHCO₃ for 15 min before the addition of 2 µm fluorescent beads (green) as in Fig. 1 E. Bar, 50 µm. (C) Bright field images of CF mouse ileal tissue mounted in horizontal chamber with charcoal added to visualize the mucus surface. 115 mM NaHCO₃ was added to the already formed mucus on CF ileal explants and incubated for 30 min. Images show mucus before and after mucus aspiration. See Video 5. (D) Transmission electron microscopic images of surface epithelia and crypts in CftrΔF508 mouse ileum (n = 2) after 15-min treatment with 115 mM NaHCO₃. Note the characteristic distension in the CF crypts. Bars, 2 µm. Arrowhead points to contact between mucus and epithelium. Data are presented as mean ± SEM.