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. 2017 Oct;5(6):887-897.
doi: 10.1177/2050640617691690. Epub 2017 Jan 29.

Downregulation of Mucosal Mast Cell Activation and Immune Response in Diarrhoea-Irritable Bowel Syndrome by Oral Disodium Cromoglycate: A Pilot Study

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Free PMC article

Downregulation of Mucosal Mast Cell Activation and Immune Response in Diarrhoea-Irritable Bowel Syndrome by Oral Disodium Cromoglycate: A Pilot Study

Beatriz Lobo et al. United European Gastroenterol J. .
Free PMC article

Abstract

Background and goal: Diarrhoea-predominant irritable bowel syndrome (IBS-D) exhibits intestinal innate immune and mucosal mast cell (MC) activation. MC stabilisers have been shown to improve IBS symptoms but the mechanism is unclear. Our primary aim was to investigate the effect of oral disodium cromoglycate (DSCG) on jejunal MC activation and specific innate immune signalling pathways in IBS-D, and secondarily, its potential clinical benefit.

Study: Mucosal MC activation (by ultrastructural changes, tryptase release and gene expression) and innate immune signalling (by protein and gene expression) were quantified in jejunal biopsies from healthy (HS; n = 16) and IBS-D subjects after six months of either treatment with DSCG (600 mg/day, IBS-D-DSCG group; n = 18) or without treatment (IBS-D-NT group; n = 25). All IBS-D patients recorded abdominal pain and bowel habits at baseline and in the last 10 days prior to jejunal sampling.

Results: IBS-D-NT exhibited significant MC activation and over-expression of immune-related genes as compared to HS, whereas in IBS-D-DSCG MC activity and gene expression were similar to HS. Furthermore, DSCG significantly reduced abdominal pain and improved stool consistency.

Conclusion: Oral DSCG modulates mucosal immune activity and improves gut symptoms in IBS-D patients. Future placebo-controlled clinical trials are needed for confirmation of clinical benefit of DSCG for IBS-D.

Keywords: Diarrhea-predominant irritable bowel syndrome; Toll-like receptors; disodium cromoglycate; intestinal innate immunity; mast cell.

Figures

Figure 1.
Figure 1.
Flowchart for intestinal samples processing. The chart indicates the number of samples subjected to the experimental procedures in each experimental group (healthy subjects, HS; IBS-D patients after six months’ follow-up with no treatment, IBS-D-NT; and after six months of treatment with oral disodium cromoglycate, IBS-D-DSCG). Intestinal fluid and a single jejunal biopsy per participant were obtained. Biopsy was immediately split into two similar pieces with a sterile scalpel. One fragment was fixed in formalin and embedded in paraffin for further microscopic examination; the remaining fragment was fixed in glutaraldehyde or in RNA stabilisation solution. Due to limited biopsy size, biopsy samples were randomised into three types of analysis (protein allocation and expression, gene expression and ultrastructure). IBS-D: diarrhoea-predominant irritable bowel syndrome; MC: mast cell; IEL: intraepithelial lymphocyte; TLR: Toll-like receptor.
Figure 2.
Figure 2.
Effect of oral disodium cromoglycate on MC activation. (a) Representative transmission electron photomicrographs of MCs in the jejunal mucosa. MCs from healthy subjects (HS), IBS-D patients after six months’ follow-up with no treatment (IBS-D-NT), and after six months of treatment with oral disodium cromoglycate (IBS-D-DSCG). Bars indicate 1 µm. Note MCs from IBS-D-NT patients displayed marked signs of piecemeal degranulation (white arrows) compared to IBS-D-DSCG patients and HS, in which a higher proportion of filled granules (black arrows) are observed. (b) Degranulation rate of individual MCs from each group was assessed according to ultrastructural characteristics of granules. Results are shown as box-and-whisker plot, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles. Groups were compared using Kruskal-Wallis test *p < 0.05, all after Dunn’s multiple comparison test *p < 0.05. Note significantly higher degranulation rates in NT patients compared to HS and DSCG. (c) mRNA expression of genes related with MC activation: tryptase (TPSAB1/B2) and chymase 1 (CMA1), assessed by Q-RT-PCR. The 18 S was used as house-keeping gene. Individual and mean values (horizontal lines) represent the fold-change against the average of the HS group in HS (n = 10), IBS-D-NT (n = 16), and IBS-D-DSCG (n = 11). Groups were compared using one-way ANOVA. *p < 0.05; after Bonferroni post-test for multiple comparison test. Note differences between IBS-D-NT patients vs IBS-D-DSCG patients and HS. MC: mast cell; mRNA: messenger RNA; Q-RT-PCR: quantitative real-time polymerase chain reaction; ANOVA: analysis of variance.
Figure 3.
Figure 3.
Mucosal expression of genes involved in innate immune activation. TLRs signalling: mRNA expression of Toll-like receptor 4 (TLR4), lymphocyte antigen (Ly96), Toll-like receptor 2 (TLR2), nucleotide-binding oligomerisation domain-containing protein 1 (NOD1), single immunoglobulin interleukin-1-related receptor (SIGIRR), and Toll interacting protein (TOLLIP). mRNA expression was assessed by Q-RT-PCR. The 18 S was utilised as house-keeping gene. Individual and mean values (horizontal lines) represent the fold-change against the average of the HS group in HS (n = 10), IBS-D-NT (n = 16), and IBS-D-DSCG (n = 11). Groups were compared using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.0001; all after Bonferroni post-test for multiple comparison test. Note differences between IBS-D-NT patients vs IBS-D-DSCG patients and HS. mRNA: messenger RNA; Q-RT-PCR: quantitative real-time polymerase chain reaction; ANOVA: analysis of variance.
Figure 4.
Figure 4.
TLR4 and tryptase protein distribution in the jejunal mucosa. Representative micrographs of double immunostaining of tryptase (green) and TLR4 (red) in the epithelium and in the lamina propria from HS, IBS-D-NT, and IBS-D-DSCG groups. Original magnification ×100. Note TLR4 (white arrows) independent of MCs (grey arrowheads). TLR4: Toll-like receptor 4; HS: healthy subjects; IBS-D-NT: diarrhoea-predominant irritable bowel syndrome patients after six months’ follow-up with no treatment; IBS-D-DSCG: diarrhoea-predominant irritable bowel syndrome patients after six months of treatment with oral disodium cromoglycate; MCs: mast cells.
Figure 5.
Figure 5.
TLR2 and tryptase protein distribution in the jejunal mucosa. Representative micrographs of double immunostaining of tryptase (green) and TLR2 (red) in the epithelium and in the lamina propria from HS, IBS-D-NT, and IBS-D-DSCG groups. Original magnification ×100. Note TLR2 expression (white arrows) independent of MCs (grey arrowheads). TLR2: Toll-like receptor 2; HS: healthy subjects; IBS-D-NT: diarrhoea-predominant irritable bowel syndrome patients after six months’ follow-up with no treatment; IBS-D-DSCG: diarrhoea-predominant irritable bowel syndrome patients after six months of treatment with oral disodium cromoglycate; MCs: mast cells.

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