Åsa V. Keita

Corticotropin releasing hormone (CRH) regulates macromolecular permeability via mast cells in normal human colonic biopsies in vitro

Objective: Persistent stress and life events affect the course of ulcerative colitis and irritable bowel syndrome by largely unknown mechanisms. Corticotropin-releasing hormone (CRH) has been implicated as an important mediator of stress-induced abnormalities in intestinal mucosal function in animal models, but to date no studies in human colon have been reported. The aim was to examine the effects of CRH on mucosal barrier function in the human colon and to elucidate the mechanisms involved in CRH-induced hyper-permeability. Design: Biopsies from 39 volunteers were assessed for macromolecular permeability (horseradish peroxidise (HRP), 51Cr-EDTA), and electrophysiology after CRH challenge in Ussing chambers. The biopsies were examined by electron and confocal microscopy for HRP and CRH receptor localisation, respectively. Moreover, CRH receptor mRNA and protein expression were examined in the human mast cell line, HMC-1. Results: Mucosal permeability to HRP was increased by CRH (2.8±0.5 pmol/cm2/h) compared to vehicle exposure (1.5±0.4 pmol/cm2/h), p = 0.032, whereas permeability to 51Cr-EDTA and transmucosal electrical resistance were unchanged. The increased permeability to HRP was abolished by α-helical CRH (9-41) (1.3±0.6 pmol/cm2/h) and the mast cell stabiliser, lodoxamide (1.6±0.6 pmol/cm2/h). Electron microscopy showed transcellular passage of HRP through colonocytes. CRH receptor subtypes R1 and R2 were detected in the HMC-1 cell line and in lamina propria mast cells in human colon. Conclusions: Our results suggest that CRH mediates transcellular uptake of HRP in human colonic mucosa via CRH receptor subtypes R1 and R2 on subepithelial mast cells. CRH-induced macromolecular uptake in human colon mucosa may have implications for stress-related intestinal disorders.

The intestinal barrier and its regulation by neuroimmune factors

Background  The ability to control uptake across the mucosa and protect from damage of harmful substances from the lumen is defined as intestinal barrier function. A disturbed barrier dysfunction has been described in many human diseases and animal models, for example, inflammatory bowel disease, irritable bowel syndrome, and intestinal hypersensitivity. In most diseases and models, alterations are seen both of the paracellular pathway, via the tight junctions, and of the transcellular routes, via different types of endocytosis. Recent studies of pathogenic mechanisms have demonstrated the important role of neuroimmune interaction with the epithelial cells in the regulation of barrier function. Neural impulses from extrinsic vagal and/or sympathetic efferent fibers or intrinsic enteric nerves influence mucosal barrier function via direct effects on epithelial cells or via interaction with immune cells. For example, by nerve‐mediated activation by corticotropin‐releasing hormone or cholinergic pathways, mucosal mast cells release a range of mediators with effects on transcellular, and/or paracellular permeability (for example, tryptase, TNF‐α, nerve growth factor, and interleukins).

Translocation of Crohn's disease Escherichia coli across M-cells: contrasting effects of soluble plant fibres and emulsifiers

Background Crohns disease is common in developed nations where the typical diet is low in fibre and high in processed food. Primary lesions overlie Peyers patches and colonic lymphoid follicles where bacterial invasion through M-cells occurs. We have assessed the effect of soluble non-starch polysaccharide (NSP) and food emulsifiers on translocation of Escherichia coli across M-cells. Methods To assess effects of soluble plant fibres and food emulsifiers on translocation of mucosa-associated E coli isolates from Crohns disease patients and from non-Crohns controls, we used M-cell monolayers, generated by co-culture of Caco2-cl1 and Raji B cells, and human Peyers patches mounted in Ussing chambers. Results E coli translocation increased across M-cells compared to parent Caco2-cl1 monocultures; 15.8-fold (IQR 6.2–32.0) for Crohns disease E coli (N=8) and 6.7-fold (IQR 3.7–21.0) for control isolates (N=5). Electron microscopy confirmed E coli within M-cells. Plantain and broccoli NSP markedly reduced E coli translocation across M-cells at 5 mg/ml (range 45.3–82.6% inhibition, p<0.01); apple and leek NSP had no significant effect. Polysorbate-80, 0.01% vol/vol, increased E coli translocation through Caco2-cl1 monolayers 59-fold (p<0.05) and, at higher concentrations, increased translocation across M-cells. Similarly, E coli translocation across human Peyers patches was reduced 45±7% by soluble plantain NSP (5 mg/ml) and increased 2-fold by polysorbate-80 (0.1% vol/vol). Conclusions Translocation of E coli across M-cells is reduced by soluble plant fibres, particularly plantain and broccoli, but increased by the emulsifier Polysorbate-80. These effects occur at relevant concentrations and may contribute to the impact of dietary factors on Crohns disease pathogenesis.

Impaired duodenal mucosal integrity and low-grade inflammation in functional dyspepsia

Objective Functional dyspepsia (FD) is an extremely common functional gastrointestinal disorder, the pathophysiology of which is poorly understood. We hypothesised that impaired intestinal barrier function is involved in the onset and persistence of this disorder by inducing low-grade inflammation. Therefore, our aim was to evaluate duodenal mucosal integrity and low-grade inflammation in patients with FD. Design Duodenal biopsy specimens were obtained from 15 patients with FD fulfilling the Rome III criteria and 15 age- and gender-matched healthy volunteers. Transepithelial electrical resistance (TEER) and paracellular permeability were measured in Ussing chambers. Expression of cell-to-cell adhesion proteins was evaluated by real-time PCR, western blot and/or immunofluorescence. Numbers of mast cells, eosinophils and intraepithelial lymphocytes were assessed by immunohistochemistry. Results Patients with FD displayed lower TEER and increased paracellular passage compared with healthy controls, which is indicative of impaired mucosal integrity. In addition, abnormal expression of cell-to-cell adhesion proteins at the level of tight junctions, adherens junctions and desmosomes was shown. Furthermore, patients were characterised by the presence of low-grade inflammation, as demonstrated by increased infiltration of mucosal mast cells and eosinophils. A significant association between the expression level of several cell-to-cell adhesion proteins, the extent of increased permeability and the severity of low-grade inflammation was found. Conclusions These findings challenge the classical paradigm that patients with FD show no structural changes in the gastrointestinal tract. We suggest that impaired intestinal barrier function is a pathophysiological mechanism in FD. Thus, restoration of intestinal barrier integrity may be a potential therapeutic target for treating patients with FD.

Increased uptake of non-pathogenic E. coli via the follicle-associated epithelium in longstanding ileal Crohn's disease

In Crohns disease (CD), inflammation is driven by luminal commensal micro‐organisms; however, mechanisms of early phases of inflammation need further clarification. The earliest observable lesions of recurrent CD are microscopic erosions at the specialized follicle‐associated epithelium (FAE), which lines the Peyers patches. Therefore, our aim was to investigate the mucosal barrier to non‐pathogenic bacteria in FAE of CD. The FAE of macroscopically normal ileum from patients with longstanding CD, ulcerative colitis, and controls was studied in Ussing chambers regarding electrophysiology and permeability to 51Cr‐EDTA, horseradish peroxidase, and non‐pathogenic E. coli strains. Transepithelial passage routes and uptake into dendritic cells were studied by confocal and electron microscopy. FAE of CD showed increased numbers of adherent bacteria, after E. coli exposure in Ussing chambers, as well as spontaneously in non‐exposed archival surgical tissues. Further, we found increased uptake of fluorescent E. coli K‐12 and HB101 across FAE of CD, but not in ulcerative colitis. Microscopy demonstrated intercellular and transcellular uptake of E. coli in CD, but only transcellular in controls. FAE exposed to E. coli demonstrated changes in conductance and 51Cr‐EDTA permeability, suggesting that bacteria affected the paracellular pathway in CD mucosa. Following bacterial uptake, CD mucosa also demonstrated an increased percentage of E. coli co‐localizing with dendritic cells, and augmented tissue release of TNF‐α. Our data present novel insights into the pathophysiology of CD by demonstrating a previously unrecognized defect of FAE barrier to bacteria in ileal CD, leading to increased load of commensal bacteria to the inductive sites of mucosal immunity. Copyright

Faecalibacterium prausnitzii supernatant improves intestinal barrier function in mice DSS colitis

Abstract Objective. The intestinal microbiota plays a substantial role in the pathogenesis of inflammatory bowel disease (IBD). Faecalibacterium prausnitzii (FP) is underrepresented in IBD patients and have been suggested to have anti-inflammatory effects in mice. Increased intestinal permeability is common in IBD but the relationship between FP and intestinal barrier function has not been investigated. Our aim was to study treatment with FP supernatant on intestinal barrier function in a dextran sodium sulfate (DSS) colitis mice model. Material and methods. C57BL/6 mice received 3% DSS in tap water ad libitum during five days to induce colitis. From day 3 the mice received a daily gavage with FP supernatant or broth during seven days. Ileum and colon were mounted in Ussing chambers for permeability studies with 51Cr-EDTA and Escherichia coli K-12. Colon was saved for Western blot analyses of tight junction proteins. Results. DSS-treated mice showed significant weight loss and colon shortening. Gavage with FP supernatant resulted in a quicker recovery after DSS treatment and less extensive colonic shortening. Ileal mucosa of DSS mice showed a significant increase in 51Cr-EDTA-passage compared to controls. 51Cr-EDTA passage was significantly decreased in mice receiving FP supernatant. No significant differences were observed in passage of E. coli K12. Western blots showed a trend to increased claudin-1 and claudin-2 expressions in DSS mice. Conclusions. Supernatant of FP enhances the intestinal barrier function by affecting paracellular permeability, and may thereby attenuate the severity of DSS-induced colitis in mice. These findings suggest a potential role of FP in the treatment of IBD.

Identification of cell adhesion molecules in the human follicle-associated epithelium that improve nanoparticle uptake into the Peyer's patches

The aim of this study was to identify cell adhesion molecules that could serve as targets of the human follicle-associated epithelium (FAE) overlying Peyers patches and to assess nanoparticle uptake levels across this epithelium. We first studied the expression of the mouse M-cell marker β1-integrin and used a model of human FAE derived from intestinal epithelial Caco-2 cells and Raji B-cells to identify additional potential targets by cDNA array. The protein expression of potential targets in the model FAE and in human ileal FAE tissues was quantified by immunofluorescence. Integrin targeting was studied by investigating the transport of Arg-Gly-Asp (RGD)-coated (integrin-binding), Arg-Gly-Glu (RGE)-coated (nonintegrin-binding), and uncoated nanoparticles across ileal specimens mounted in Ussing chambers. Both β1-integrin and the cell adhesion molecule CD9 were more abundantly expressed in the model and human FAE compared with the Caco-2 control cells or villus epithelium (VE). Uncoated nanoparticles were not taken up across either FAE or VE. General integrin targeting with RGD improved the nanoparticle transport dramatically across the FAE and to a lower extent across the VE. Compared with RGE, RGD improved transport 4-fold across the FAE. There was no difference in the transport of RGD- and RGE-coated nanoparticles across the VE. In conclusion, β1-integrin and CD9 were identified as targets in human FAE. The difference in RGD- and RGE-mediated transport across the FAE, but not the VE, suggests that a specific integrin interaction was the dominating mechanism for improved nanoparticle uptake across the FAE., whereas charge interaction contributed substantially to the improved VE uptake.

Characterization of antigen and bacterial transport in the follicle-associated epithelium of human ileum.

The follicle-associated epithelium (FAE), covering Peyers patches, provides a route of entry for antigens and microorganisms. Animal studies showed enhanced antigen and bacterial uptake in FAE, but no study on barrier function of human FAE has been reported. Our aim was to characterize the normal barrier properties of human FAE. Specimens of normal ileum were taken from 30 patients with noninflammatory colonic disease. Villus epithelium (VE) and FAE were identified and mounted in Ussing chambers. Permeability to 51Cr-EDTA, transmucosal flux of the protein antigen, horseradish peroxidase (HRP), and transport of fluorescent Escherichia coli (chemically killed K-12 and live HB101) were measured. Uptake mechanisms were studied by confocal- and transmission electron microscopy, and by using pharmacological inhibitors in an in vitro coculture model of FAE and in human ileal FAE. HRP flux was substantially higher in FAE than in VE, and was reduced by an amiloride analog. Electron microscopy showed HRP-containing endosomes. Transport of E. coli K-12 and HB101 was also augmented in FAE and was confirmed by confocal microscopy. In vitro coculture experiments and electron microscopy revealed actin-dependent, mainly transcellular, uptake of E. coli K-12 into FAE. 51Cr-EDTA permeability was equal in FAE and VE. Augmented HRP flux and bacterial uptake but similar paracellular permeability, suggest functional variations of transcellular transport in the FAE. We show for the first time that FAE of human ileum is functionally distinct from regular VE, rendering the FAE more prone to bacterial–epithelial cell interactions and delivery of antigens to the mucosal immune system.

CD83+CCR7− Dendritic Cells Accumulate in the Subepithelial Dome and Internalize Translocated Escherichia coli HB101 in the Peyer’s Patches of Ileal Crohn’s Disease

Recurrent Crohns disease originates with small erosions in the follicle-associated epithelium overlying the Peyers patches. Animal studies have illustrated mucosal immune regulation by dendritic cells located in the subepithelial dome. The aim of this study was to characterize the dendritic cells at this specific site in patients with Crohns disease. Ileal tissues were obtained after surgery performed on Crohns patients; ileal samples from noninflammatory bowel disease and ulcerative colitis served as standard and inflammatory controls, respectively. Flow cytometry of isolated intestinal mononuclear cells showed a larger subset of dendritic cells in Crohns samples compared with controls. This finding was corroborated by confocal microscopy, showing enhanced infiltrates of cells positive for the dendritic cell markers, DC-SIGN(+) and CD83(+), in the subepithelial dome. Moreover, the CD83(+) cells in Crohns tissues showed reduced expression of the lymph node migratory receptor, CCR7, possibly contributing to the high numbers of dendritic cells. After exposure to nonpathogenic Escherichia coli in Ussing chambers, dendritic cells in the subepithelial dome of Crohns disease demonstrated increased co-localization with translocated bacteria. Immunohistochemical results revealed that DC-SIGN(+) cells in Crohns tissues were found to express toll-like receptor 4 and produce tumor necrosis factor-alpha. In conclusion, nonmigrating dendritic cells that accumulate in the subepithelial dome and internalize nonpathogenic bacteria may be important for the onset and perpetuation of mucosal inflammation in Crohns disease.

Stress-induced barrier disruption of rat follicle-associated epithelium involves corticotropin-releasing hormone, acetylcholine, substance P, and mast cells

Background  The follicle‐associated epithelium (FAE) is specialized in uptake and sampling of luminal antigens and bacteria. We previously showed that stress increased FAE permeability in rats. An increased uptake may alter antigen exposure in Peyer’s patches leading to intestinal disease. The aim of this study was to elucidate mechanisms involved in the acute stress‐induced increase in FAE permeability.