Tricellulin is regulated via interleukin-13-receptor α2, affects macromolecule uptake, and is decreased in ulcerative colitis.

In the two inflammatory bowel diseases, ulcerative colitis (UC) and Crohn's disease (CD), altered expression of tight junction (TJ) proteins leads to an impaired epithelial barrier including increased uptake of luminal antigens supporting the inflammation. Here, we focused on regulation of tricellulin (Tric), a protein of the tricellular TJ essential for the barrier against macromolecules, and hypothesized a role in paracellular antigen uptake. We report that Tric is downregulated in UC, but not in CD, and that its reduction increases the passage of macromolecules. Using a novel visualization method, passage sites were identified at TJ regions usually sealed by Tric. We show that interleukin-13 (IL-13), beyond its known effect on claudin-2, downregulates Tric expression. These two effects of IL-13 are regulated by different signaling pathways: The IL-13 receptor α1 upregulates claudin-2, whereas IL-13 receptor α2 downregulates Tric. We suggest to target the α2 receptor in future developments of therapeutical IL-13-based biologicals.

TcpC protein from E. coli Nissle improves epithelial barrier function involving PKCζ and ERK1/2 signaling in HT-29/B6 cells.

The probiotic Escherichia coli Nissle 1917 (EcN) is widely used to maintain remission in ulcerative colitis. This is thought to be mediated by various immunomodulatory and barrier-stabilizing effects in the intestine. In this study, the mechanisms of barrier modulation by EcN were studied in the human epithelial HT-29/B6 cell culture model.EcN supernatant increased transepithelial resistance (TER) and reduced permeability to mannitol because of sealing of the paracellular passage pathway as revealed by two-path impedance spectroscopy. This increase in TER was attributed to the TcpC protein of EcN. TcpC induced protein kinase C-ζ (PKCζ) and extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation, which in turn resulted in upregulation of the barrier-forming tight junction protein claudin-14. By specific silencing of protein expression by small interfering RNA (siRNA), the sealing function of claudin-14 was confirmed. In conclusion, the TcpC protein of EcN affects innate immunity by improving intestinal barrier function through upregulation of claudin-14 via PKCζ and ERK1/2 signaling.

Cross-talk Between Host, Microbiome and Probiotics: A Systems Biology Approach for Analyzing the Effects of Probiotic Enterococcus faecium NCIMB 10415 in Piglets.

A comprehensive data-set from a multidisciplinary feeding experiment with the probiotic Enterococcus faecium was analyzed to elucidate effects of the probiotic on growing piglets. Sixty-two piglets were randomly assigned to a control (no probiotic treatment) and a treatment group (E. faecium supplementation). Piglets were weaned at 26 d. Age-matched piglets were sacrificed for the collection of tissue samples at 12, 26, 34 and 54 d. In addition to zootechnical data, the composition and activity of intestinal microbiota, immune cell types, and intestinal responses were determined. Our systems analysis revealed clear effects on several measured variables in 26 and 34 days old animals, while response patterns varied between piglets from different age groups. Correlation analyses identified reduced associations between intestinal microbial communities and immune system reactions in the probiotic group. In conclusion, the developed model is useful for comparative analyses to unravel systems effects of dietary components and their time resolution. The model identified that effects of E. faecium supplementation most prominently affected the interplay between intestinal microbiota and the intestinal immune system. These effects, as well as effects in other subsystems, clustered around weaning, which is the age where piglets are most prone to diarrhea.

Enterococcus faecium NCIMB 10415 does not protect interleukin-10 knock-out mice from chronic gut inflammation.

Enterococcus faecium NCIMB 10415 reduces diarrhoea incidence and duration in animals and human study subjects. We tested whether the strain is also capable of reducing chronic gut inflammation and aimed to identify mechanisms that are involved in possible probiotic effects. To identify health-promoting mechanisms of the strain, we used interleukin-10-deficient mice that spontaneously develop gut inflammation and fed these mice a diet containing NCIMB 10415 for 3, 8 and 24 weeks, respectively. Control mice were fed a diet which was identically composed but did not contain the strain. After 3 weeks of intervention the experimental animals were less inflamed in the caecum than the control animals. This effect was not observed in the colon and there were no differences between experimental and control mice at any other time point. The application of the strain was associated with higher expression levels of interferon gamma and interferon gamma-induced protein 10 after 3 and 24 but not after 8 weeks of feeding. No differences between the animals were observed in intestinal barrier function or intestinal microbiota composition. However, we observed a low abundance of the mucin-degrading bacterium Akkermansia muciniphila in the mice that were fed NCIMB 10415 for 8 weeks. These low cell numbers were associated with a significantly lower caecal inflammation score and improved paracellular permeability as compared to the NCIMB-treated mice that were killed after 3 and 24 weeks of intervention. In conclusion, NCIMB 10415 is not capable of reducing gut inflammation in the IL-10-/- mouse model. The exact role of A. muciniphila and of a possible interaction between this bacterium, NCIMB 10415 and the host in gut inflammation requires further investigation.

Influence of fermentable carbohydrates or protein on large intestinal and urinary metabolomic profiles in piglets.

It was recently shown that variations in the ratio of dietary fermentable carbohydrates (fCHO) and fermentable protein (fCP) differentially affect large intestinal microbial ecology and the mucosal response. Here we investigated the use of mass spectrometry to profile changes in metabolite composition in colon and urine associated with variation in dietary fCHO and fCP composition and mucosal physiology. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP and low fCHO, low fCP and high fCHO, high fCP and low fCHO, and high fCP and high fCHO. After 21 to 23 d, all pigs were euthanized and colon digesta and urine metabolite profiles were obtained by mass spectrometry. Analysis of mass spectra by partial least squares approach indicated a clustering of both colonic and urinary profiles for each pig by feeding group. Metabolite identification and annotation using the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways revealed increased abundance of metabolites associated with arachidonic acid metabolism in colon of pigs fed a high concentration of fCP irrespective of dietary fCHO. Urinary metabolites did not show as clear patterns. Mass spectrometry can effectively differentiate metabolite profiles in colon contents and urine associated with changes in dietary composition. Whether metabolite profiling is an effective tool to identify specific metabolites (biomarkers) or metabolite profiles associated with gut function and integrity needs further elucidation.

Mechanisms of epithelial translocation of the alpha(2)-gliadin-33mer in coeliac sprue.

BACKGROUND AND AIMS: The alpha(2)-gliadin-33mer has been shown to be important in the pathogenesis of coeliac disease. We aimed to study mechanisms of its epithelial translocation and processing in respect to transcytotic and paracellular pathways. METHODS: Transepithelial passage of a fluorescence-labelled alpha(2)-gliadin-33mer was studied in Caco-2 cells by using reverse-phase high-performance liquid chromatography, mass spectrometry, confocal laser scanning microscopy (LSM) and fluorescence activated cell sorting (FACS). Endocytosis mechanisms were characterised with rab-GFP constructs transiently transfected into Caco-2 cells and in human duodenal biopsy specimens. RESULTS: The alpha(2)-gliadin-33mer dose-dependently crossed the epithelial barrier in the apical-to-basal direction. Degradation analysis revealed translocation of the 33mer polypeptide in the uncleaved as well as in the degraded form. Transcellular passage was identified by confocal LSM, inhibitor experiments and FACS. Rab5 but not rab4 or rab7 vesicles were shown to be part of the transcytotic pathway. After pre-incubation with interferon-gamma, translocation of the 33mer was increased by 40%. In mucosal biopsies of the duodenum, epithelial 33mer uptake was significantly higher in untreated coeliac disease patients than in healthy controls or coeliac disease patients on a gluten-free diet. CONCLUSION: Epithelial translocation of the alpha(2)-gliadin-33mer occurs by transcytosis after partial degradation through a rab5 endocytosis compartment and is regulated by interferon-gamma. Uptake of the 33mer is higher in untreated coeliac disease than in controls and coeliac disease patients on a gluten-free diet.

Restitution of single-cell defects in the mouse colon epithelium differs from that of cultured cells.

Integrity of colon epithelium is of crucial importance and, as small defects occur constantly, rapid repair (restitution) is essential. To investigate the mechanism of restitution, single-cell lesions were induced in mouse colonic surface epithelia by iontophoretic injection of Ca2+. Closure of the resulting defects was monitored using confocal laser scanning microscopy (CLSM), and functional sealing by electrophysiological techniques. Restitution was evaluated as the time constant tau of the exponential decrease in conductance of an induced leak and amounted to 0.28 min under control conditions. After 4 min, the leak was completely sealed. Repair was thus considerably faster than in previously investigated HT-29/B6 cells (tau=5.73 min). As in cultured cells, cytochalasin D delayed restitution in native colon epithelia (tau=0.69 min), indicating the involvement of actin in the healing process; however, no accumulation of actin surrounding the lesion was detected. Long-term incubation of epithelia with IFN-gamma alone or in combination with TNF-alpha increased tau to 0.49 and 0.59 min, respectively. In contrast to cultured cells, TNF-alpha alone did not affect restitution. A brief (<10 min) exposure to the sterile filtered supernatant of hemolytic E. coli O4 cultures did not affect the morphology of the epithelium, but delayed restitution. In CLSM studies, defects were still clearly visible 4 min after the onset of lesion induction. The supernatant of a nonhemolytic E. coli O4 mutant did not exhibit this effect. In conclusion, single-cell defects in native colon cause functional leaks that seal faster than in cell cultures. Proinflammatory cytokines and pathogenic bacteria delay restitution. This suggests a key role of very small lesions at the onset of pathogenic processes in the intestine.