A β-Glucan-Based Dietary Fiber Reduces Mast Cell-Induced Hyperpermeability in Ileum From Patients With Crohn's Disease and Control Subjects

John-Peter Ganda Mall, Maite Casado-Bedmar, Martin E Winberg, Robert J Brummer, Ida Schoultz, Åsa V Keita, John-Peter Ganda Mall, Maite Casado-Bedmar, Martin E Winberg, Robert J Brummer, Ida Schoultz, Åsa V Keita

Abstract

Background: Administration of β-glucan has shown immune-enhancing effects. Our aim was to investigate whether β-glucan could attenuate mast cell (MC)-induced hyperpermeability in follicle-associated epithelium (FAE) and villus epithelium (VE) of patients with Crohn's disease (CD) and in noninflammatory bowel disease (IBD)-controls. Further, we studied mechanisms of β-glucan uptake and effects on MCs in vitro.

Methods: Segments of FAE and VE from 8 CD patients and 9 controls were mounted in Ussing chambers. Effects of the MC-degranulator compound 48/80 (C48/80) and yeast-derived β-1,3/1,6 glucan on hyperpermeability were investigated. Translocation of β-glucan and colocalization with immune cells were studied by immunofluorescence. Caco-2-cl1- and FAE-cultures were used to investigate β-glucan-uptake using endocytosis inhibitors and HMC-1.1 to study effects on MCs.

Results: β-glucan significantly attenuated MC-induced paracellular hyperpermeability in CD and controls. Transcellular hyperpermeability was only significantly attenuated in VE. Baseline paracellular permeability was higher in FAE than VE in both groups, P<0.05, and exhibited a more pronounced effect by C48/80 and β-glucan P<0.05. No difference was observed between CD and controls. In vitro studies showed increased passage, P<0.05, of β-glucan through FAE-culture compared to Caco-2-cl1. Passage was mildly attenuated by the inhibitor methyl-β-cyclodextrin. HMC-1.1 experiments showed a trend to decreasing MC-degranulation and levels of TNF-α but not IL-6 by β-glucan. Immunofluorescence revealed more β-glucan-uptake and higher percentage of macrophages and dendritic cells close to β-glucan in VE of CD compared to controls.

Conclusions: We demonstrated beneficial effects of β-glucan on intestinal barrier function and increased β-glucan-passage through FAE model. Our results provide important and novel knowledge on possible applications of β-glucan in health disorders and diseases characterized by intestinal barrier dysfunction.

Keywords: Crohn’s disease; intestinal permeability; β-glucan.

© 2017 Crohn’s & Colitis Foundation of America. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Figures

FIGURE 1.
FIGURE 1.
Effects of yeast-derived β-glucan on compound 48/80 (C48/80)-induced paracellular hyperpermeability in VE and FAE of 8 control subjects mounted in Ussing chambers. (A-B) Control subjects displayed an increased FITC-dextran passage in VE and FAE after stimulation with the mast cell degranulator C48/80 compared to unstimulated tissues (vehicles). Costimulation with β-glucan attenuated C48/80 effects to levels close to vehicles. A similar pattern was seen for CD patients (Suppl Fig. 1). Data (∆90—0 min) are presented as a line intersecting the median and each dot representing one patient, ***P < 0.001
FIGURE 2.
FIGURE 2.
Effects of yeast-derived β-glucan on compound 48/80 (C48/80)-induced transcellular hyperpermeability in VE and FAE of 6 control subjects mounted in Ussing chambers. (A-B) Control subjects displayed an increased HRP passage in VE after stimulation with the mast cell degranulator C48/80 compared to unstimulated VE (vehicle). Costimulation with β-glucan attenuated C48/80 effects to levels close to vehicle. In FAE, there was a significant increase in HRP passage after C48/80 stimulation, however, costimulation with β-glucan showed no effect. A similar pattern was seen for CD patients, although lacking significance in FAE (Suppl Fig. 2). Data (∆90—0 min) are presented as a line intersecting the median and each dot representing one patient. *P < 0.05, ***P < 0.001
FIGURE 3.
FIGURE 3.
Uptake of Alexa Fluor 594-conjugated yeast-derived β-glucan into the VE and FAE of patients with CD and control subjects, after 20 minutes in Ussing chambers. (A) Uptake of β-glucan (red, arows) in the VE of a control subject. (B) Uptake of β-glucan (red, arrows) in the VE of a CD patient (C) Uptake of β-glucan (red, arrows) in the FAE of a CD patient. Photographs show β-glucan close to the epithelial cell lining as well as further down in the underlying lamina propria/subepithelial dome and follicle. Photographs are in 200X magnification. (D) Quantification of β-glucan uptake. Values are presented as median (25th-75th percentile) and comparisons between groups were done with Mann- Whitney U test, *P < 0.05
FIGURE 4.
FIGURE 4.
Fluorescence microscopy of Alexa Fluor 594-conjugated yeast-derived β-glucan translocating through the VE and FAE from patients with CD and control subjects. After 20 minutes in Ussing chambers, tissues were stained for MC tryptase or CD68. (A) Staining of MCs (green) in Peyer’s patches from a CD patient showing β-glucan (red, arrows) close to the FAE (1), within the adjacent villi to the FAE (2) and occasionally colocalizing with MCs . (B) Staining of MCs (green) in VE of a CD patient showing β-glucan (arrows) in close proximity to MCs (1–2). (C) Staining of CD68 in VE from a control patient showing β-glucan (red, arrows) in close proximity to macrophages (green) (1- 2), or in a direct colocalization with macrophages (2, arrowhead). Overview photographs are in 400X magnification and zoomed pictures in 1000X
FIGURE 5.
FIGURE 5.
Fluorescence microscopy of Alexa Fluor 594-conjugated yeast-derived β-glucan translocating through the FAE and underlying Peyer’s patches from a patient with CD. After 20 minutes in Ussing chambers, tissue was stained for the DC marker DC-SIGN. Microscopy revealed DCsDC-SIGN+ (green) in the subepithelial dome and further down in the Peyer’s patches at follicle margins in close proximity to β-glucan (1–3, red, arrows). Windows 1 and 3 illustrate a direct colocalization between β-glucan and DCsDC-SIGN+ (arrowheads). Overview photographs are in 400X magnification and zoomed pictures in 1000X
FIGURE 6.
FIGURE 6.
Passage and translocation of Alexa Fluor 594-conjugated yeast-derived β-glucan through monolayers of Caco-2-cl1 and FAE coculture model after 1 hour incubation. (A) β-glucan passage through the in vitro models (7 independent experiments with duplicates). (B) Intracellular β-glucan quantification by confocal microscopy of in vitro models (3 independent experiments with duplicates, 7 Z-stack areas each). (C) Representative photograph of cells stained with phalloidin (green) and translocating β-glucan (red) in the Caco2-cl1 and FAE coculture model, 63X magnification. (D-E) β-glucan passage through Caco2-cl1 and FAE model after 20 minutes incubation (D) and 1 hour incubaction (E) with and without 20 minutes preincubation with the lipid raft inhibitor MβCD (4 independent experiments with duplicates). *P < 0.05. All data (A-E) were analyzed on log2-scale with FAE model compared to Caco2-cl1 (A-B) and the MβCD-treated groups (D-E) compared to the untreated groups of the Caco-2-cl1/FAE model
FIGURE 7.
FIGURE 7.
Effects of compound 48/80 (C48/80) and yeast-derived β-glucan on degranulation and cytokine secretion of the human mast cell line HMC-1.1. Degranulation was measured by the release of β-hexosaminidase, TNF-α and IL-6 after 20 minutes of preincubation with β-glucan and 24 hours incubation with C48/80. (A) Stimulation with C48/80 significantly increased β-hexosaminidase release compared to vehicle. Preincubation with β-glucan showed a nonsignificant trend towards decreased degranulation compared to cells stimulated only with C48/80. Results were based on 9 independent experiments with duplicates. (B) Illustration of MC tryptase staining (green) of HMC-1.1 in unstimulated cells (vehicle), cells stimulated with C48/80 and cells incubated with β-glucan (red) prior C48/80-stimulation. The zoomed photograph in the lower right corner illustrates β-glucan in close proximity to a MC, visualized by DAPI nucleus staining (blue). Overview photographs are in 600X magnification and zoomed photograph in 1000X. (C) Stimulation with C48/80 gave no significantly increased levels of TNF-α, however, 20 minutes of preincubation with β-glucan before administration of C48/80 resulted in significantly lower levels of TNF-α compared to C48/80 stimulation only. (D) Stimulation with C48/80 resulted in significantly higher levels of IL-6 compared to vehicle, however, prestimulation with β-glucan had no effect on C48/80-induced degranulation. ***P < 0.001, *P < 0.05. All data were analyzed on log2-scale

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