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 2

Dynamics of goblet cell exocytosis induced by 100 μM CCH in WT and Cftr-KO enteroids.

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Dynamics of goblet cell exocytosis induced by 100 μM CCH in WT and Cftr-...
(A) Luminal superfusion of enteroid using a micropipette. Basolateral superfusate flow is from right to left. Scale bar: 50 μm. (B) Dynamics of CCH-stimulated exocytosis: 0, granule area immediately prior to CCH (basal); 1, time point when maximal granule area attained after CCH (max); 2, granule area at first visible evidence of exocytosis; 3, time point of minimal granule area attained after exocytosis (min). Lines between points do not represent data, but are provided to illustrate the continuous nature of the exocytotic process and a visual estimate of rates of change in granule area between basal→maximum and exocytosis→minimum. *P < 0.05, time period between 1→2 is significantly longer than in WT by t test. (C) Statistical analyses of cross-sectional area of granule cluster at basal (time point 0), maximum (time point 1), and minimum (time point 3). Mucus bleb area is equal to cross-sectional area of luminal bleb from a perimeter line drawn between the apical membranes of adjacent enterocytes and outlining the mass in the crypt lumen after CCH-induced exocytosis. †,‡,§Within group, means with different symbols are statistically different by ANOVA with post-hoc Tukey’s test. *P < 0.05, significantly different from WT by t test. (D) Rate of change in granule area between basal and maximum (time points 0→1) and between exocytosis and minimum (time points 2→3) after CCH exposure. *P < 0.05, significantly different from WT by t test. n = 8–14 goblet cells in enteroids from 4 WT and Cftr-KO sex-matched littermate pairs.