Vasoactive Intestinal Polypeptide and Mast Cells Regulate Increased Passage of Colonic Bacteria in Patients With Irritable Bowel Syndrome

Abstract

Background & aims: Irritable bowel syndrome (IBS) is associated with intestinal dysbiosis and symptoms of IBS develop following gastroenteritis. We aimed to study the passage of live bacteria through the colonic epithelium, and determine the role of mast cells (MCs) and vasoactive intestinal polypeptide (VIP) in barrier regulation in IBS and healthy individuals.

Methods: Colon biopsies from 32 women with IBS and 15 age-matched healthy women (controls) were mounted in Ussing chambers; we measured numbers of fluorescently labeled Escherichia coli HS and Salmonella typhimurium that passed through from the mucosal side to the serosal side of the tissue. Some biopsies were exposed to agents that block the VIP receptors (VPAC1 and VPAC2) or MCs. Levels of VIP and tryptase were measured in plasma and biopsy lysates. Number of MCs and MCs that express VIP or VIP receptors were quantified by immunofluorescence. Biopsies from an additional 5 patients with IBS and 4 controls were mounted in chambers and Salmonella were added; we studied passage routes through the epithelium by transmission electron microscopy and expression of tight junctions by confocal microscopy.

Results: In colon biopsies from patients with IBS, larger numbers of E coli HS and S typhimurium passed through the epithelium than in biopsies from controls (P < .0005). In transmission electron microscopy analyses, bacteria were found to cross the epithelium via only the transcellular route. Bacterial passage was reduced in biopsies from patients with IBS and controls after addition of antibodies against VPACs or ketotifen, which inhibits MCs. Plasma samples from patients with IBS had higher levels of VIP than plasma samples from controls. Biopsies from patients with IBS had higher levels of tryptase, larger numbers of MCs, and a higher percentage of MCs that express VPAC1 than biopsies from controls. In biopsies from patients with IBS, addition of Salmonella significantly reduced levels of occludin; subsequent addition of ketotifen significantly reversed this effect.

Conclusions: We found that colonic epithelium tissues from patients with IBS have increased translocation of commensal and pathogenic live bacteria compared with controls. The mechanisms of increased translocation include MCs and VIP.

Keywords: Bacteria; Inflammation; Intestinal Permeability; Ketotifen.

Fig. 1

Bacterial passage through colonic biopsies from 32 patients irritable bowel syndrome (IBS) and 15 healthy controls (HC) mounted in Ussing chambers for 120 min. (A) Passage of live E. coli HS and Salmonella typhimurium. Comparisons were done with Mann-Whitney U test, ^***p<0.0005 vs. HC. (B). Representative scanning electron micrographs of E. coli and Salmonella on the surface of the colonic epithelium and representative confocal micrographs illustrating bacterial uptake (white arrows) into the mucosa of IBS colon, after incubation. (C) Transmission electron micrographs illustrating interaction of Salmonella (black arrows) and microvilli (white arrows); a transversal section where interaction is shown; and bacterial uptake through endocytosis (note double membrane) at the apical side of the epithelium. (D) Intestinal bacteria quantified by Giemsa staining. Arrows indicate two bacteria just entering the epithelium in an IBS sample.

Fig. 2

Influence of vasoactive intestinal polypeptide (VIP) and mast cells (MCs) on bacterial passage through colonic biopsies from 32 patients with irritable bowel syndrome (IBS) and 15 healthy controls (HC) mounted in Ussing chambers. Figure indicates passage at 120 min of E. coli HS (A) and Salmonella typhimurium (B) before (vehicle) and after addition of VIP-receptor blocker (anti-VPACs) or MC-stabilizer (ketotifen) to the chambers. Comparisons were done with Mann-Whitney U test, ^**p<0.005, ^***p<0.0005 vs. vehicle.

Fig 3

The effect of live Salmonella typhimurium on occludin. Photographs illustrate staining for occludin in colonic biopsies from a patient with irritable bowel syndrome mounted in Ussing chambers. Biopsies were added buffer only (vehicle), Salmonella, Salmonella+vasoactive intestinal polypeptide (VIP)-receptor blocker (anti-VPACs) or Salmonella+ketotifen. After 30 min, biopsies were removed and stained for occludin by immunofluorescence.

Fig 4

Staining of mast cell (MC)-tryptase (red) and vasoactive intestinal polypeptide receptors (VPACs) (green) in colon of a patient with irritable bowel syndrome (IBS) and healthy control (HC). Photographs show MCs±VPAC1 expressions in an IBS patient (A) and HC (B). Arrows indicate individual MC or VPAC1 staining and arrowheads indicate co-localization i.e. MCs expressing VPAC1 (C) Photograph showing MCs expressing VPAC2 (arrowheads) in IBS colon. Arrows indicate individual MC or VPAC2 staining. Overview photographs; 400Xmagnification, zoomed photographs; 1000X.

Fig 5

Staining of mast cell (MC)-tryptase (red) and vasoactive intestinal polypeptide (VIP) (green) in colonic biopsy of a patient with irritable bowel syndrome (IBS). Photograph shows a MC expressing VIP (arrowhead). Window in upper right corner shows a MC in close proximity to secreted VIP, which was a common feature seen in IBS patients, but could also be observed in healthy controls. Overview photographs; 400Xmagnification, zoomed photographs; 1000X.

Fig 6

Mucosal mast cell (MC) activation: tryptase, ultrastructure and degranulation in irritable bowel syndrome (IBS) and healthy control (HC) subjects. (A) Expression of MC-tryptase (mw 31–36 kDa) and β-actin as loading control. Lysates from colonic biopsies of 32 women with IBS and 15 HCs were analyzed by Western Blot. Panels show a representative blot from 5 IBS subjects and 5 HCs. (B) Degranulation of MCs at baseline of IBS (n=19) and HC (n=8) (C) Degranulation of MC in biopsies from IBS patients (n=5) and HCs (n=4) after incubation with buffer only (vehicle), Salmonella, Salmonella+vasoactive intestinal polypeptide (VIP) receptor blocker (anti-VPACs), or Salmonella+ketotifen. (D) Micrographs of MCs from IBS and HC from the different experimental groups. Note signs of MC-activation such as irregular plasma membrane with emission of pseudopodes (black arrow) and piecemeal degranulation (black arrow head). Fusions of granules are predominantly detected in MCs from the IBS-group (white arrow).