Defective goblet cell exocytosis contributes to murine cystic fibrosis–associated intestinal disease
Jinghua Liu, Nancy M. Walker, Akifumi Ootani, Ashlee M. Strubberg, Lane L. Clarke
Jinghua Liu, Nancy M. Walker, Akifumi Ootani, Ashlee M. Strubberg, Lane L. Clarke
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Research Article Gastroenterology

Defective goblet cell exocytosis contributes to murine cystic fibrosis–associated intestinal disease

Abstract

Cystic fibrosis (CF) intestinal disease is associated with the pathological manifestation mucoviscidosis, which is the secretion of tenacious, viscid mucus that plugs ducts and glands of epithelial-lined organs. Goblet cells are the principal cell type involved in exocytosis of mucin granules; however, little is known about the exocytotic process of goblet cells in the CF intestine. Using intestinal organoids from a CF mouse model, we determined that CF goblet cells have altered exocytotic dynamics, which involved intrathecal granule swelling that was abruptly followed by incomplete release of partially decondensated mucus. Some CF goblet cells exhibited an ectopic granule location and distorted cellular morphology, a phenotype that is consistent with retrograde intracellular granule movement during exocytosis. Increasing the luminal concentration of bicarbonate, which mimics CF transmembrane conductance regulator–mediated anion secretion, increased spontaneous degranulation in WT goblet cells and improved exocytotic dynamics in CF goblet cells; however, there was still an apparent incoordination between granule decondensation and exocytosis in the CF goblet cells. Compared with those within WT goblet cells, mucin granules within CF goblet cells had an alkaline pH, which may adversely affect the polyionic composition of the mucins. Together, these findings indicate that goblet cell dysfunction is an epithelial-autonomous defect in the CF intestine that likely contributes to the pathology of mucoviscidosis and the intestinal manifestations of obstruction and inflammation.

Authors

Jinghua Liu, Nancy M. Walker, Akifumi Ootani, Ashlee M. Strubberg, Lane L. Clarke

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Figure 6

Alkaline mucin granules in goblet cells of Cftr-KO enteroids.

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Alkaline mucin granules in goblet cells of Cftr-KO enteroids. (A) Left p...
(A) Left panel, LysoSensor-stained granule cluster in a single goblet cell. Right panel, virtual measurement spots (yellow) placed on granules (green) at apical, middle, and basal thirds of granule cluster. Scale bar: 3 μm. (B) The 490λ/550λ emission ratio of LysoSensor-stained granules at the apical, middle, and basal thirds of granule mass in intact WT and Cftr-KO enteroids. Enteroids were superfused with 25 mM HCO3– KBR. *P < 0.05, significantly different from WT by t test; +P < 0.05, significantly different from ratio at basal third by repeated-measures ANOVA with post-hoc Bonferroni’s test. n = 13–14 goblet cells in intact enteroids from 6 WT and Cftr-KO sex-matched littermates. (C) The 490λ/550λ emission ratio of LysoSensor-stained granule cluster in goblet cells of bisected WT and Cftr-KO enteroids. *P < 0.05, significantly different from WT by t test. n = 6 goblet cells in bisected enteroids from 3 WT and Cftr-KO sex-matched littermate pairs. (D) Method for pHi measurement using SNARF-5F (red) and quinacrine (green) in intact WT and Cftr-KO enteroids. Arrow, virtual measurement spheres placed within the plasmalemma confines of 3D constructed goblet cells and adjacent enterocytes. Scale bar: 15 μm. (E) Mean pHi of goblet cells (GC) and adjacent enterocytes (neighbor) in intact WT and Cftr-KO enteroids. †,‡Within group, means with different symbols are statistically different by ANOVA with post-hoc Tukey’s test, P < 0.05. n = 14–17 goblet cells in enteroids from 6 WT and Cftr-KO sex-matched littermates.