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BACKGROUND: The beneficial effects of dietary fibre have been recognized for many years. These complex polysaccharides are indigestible and fermented by gut microbiota, promoting advantageous effects to intestinal mucosa indirectly. Polysaccharides also have direct effects on intestinal epithelial barrier function and we have previous shown that rhamnogalacturonan (RGal), a polysaccharide isolated from the plant Acmella oleracea, promotes barrier function in human colonic epithelial Caco-2 cells. However, the underlying mechanism of action of RGal remains unclear. AIMS: We aimed to investigate if RGal directly enhances intestinal epithelial barrier function through TLR4 activation. METHODS: Human TLR4/CD14/MD2 transfected HEK 293 reporter cells were incubated with RGal (1000 μg/ml) with/without C34 (TLR4 inhibitor, 10 ng/ml) for 24 hrs. Supernatants were collected and activation of TLR4 was detected by using the secreted alkaline phosphatase assay (Invivogen). LPS (10 ng/ml) was used as positive control. Caco-2 cells were seeded on Millicell supports for 6 days and treated apically with vehicle (0.01% DMSO in PBS) and C34 (100 ng/ml). After 1 hr, cells were mounted in Ussing chambers and treated apically with RGal (1000 μg/ml). Transepithelial electrical resistance (TER) and FITC-dextran flux were measured. In another set of experiments, the monolayers were treated apically with GF109203X (pan-PKC inhibitor, 500 nM), myristoylated PKCζ pseudosubstrate inhibitor (10 μM) and Go6976 (PKCα/β inhibitor, 10 nM) for 10 minutes before apical addition of RGal (1000 μg/ml). RESULTS: Incubation of TLR4-expressing HEK cells with RGal (1000 μg/ml) activated TLR4 at similar levels compared with LPS (10 ng/ml). In addition, C34 (10 ng/ml) significantly inhibited both RGal and LPS activation without altering cell viability (assessed via MTT assay). Apical treatment of cells with RGal increased TER and reduced FITC-dextran flux compared to control. C34 reversed the RGal effects in TER. The RGal-mediated decrease in macromolecular permeability was dependent on PKC but not on PKCζ or PKCα/β. CONCLUSIONS: These data suggest that RGal enhances intestinal epithelial barrier function through activation of TLR4 and the PKC signaling pathway. Elucidation of RGal mechanisms of action could lead to new approaches to the treatment of inflammatory bowel diseases. FUNDING AGENCIES: NSERC

BACKGROUND: It is well known that diet profoundly affects intestinal physiology, with some diets contributing to a “leaky gut”, possibly promoting IBD and metabolic syndrome. Recently, several studies have suggested that the enteric nervous system, composed of neurons and glial cells, could be a potent regulator of intestinal permeability. We hypothesize that nutrients, sensed at the level of the epithelium, could trigger a neuroglial response to regulate intestinal permeability. AIMS: We investigated whether nutrients acutely modulate intestinal permeability at the level of the small intestine and if the enteric nervous system plays a role in this effect. METHODS: Intact jejunum and Ileum segments from CD1 mice were mounted in Ussing chambers. Transepithelial electrical resistance (TER) and short circuit current (Isc) were recorded for 50 min after stimulation with different nutrients. Two different size probes, 400 Da Fluorescein isothiocyanate (FITC) or 4000 Da FITC-Dextran (FD4), were used as markers of intestinal permeability. Apical glucose, fructose, glutamine (10 mM) or 5% Intralipid® were used as nutrient stimuli. Enteric neurons were either inhibited with tetrodotoxin (TTX, 0.5 µM) or activated with veratridine (10 µM). Enteric gliotransmission was inhibited with the connexin 43 blocker Gap26 (20 µM). RESULTS: The nutrients used in this study were “sensed” by the intestinal epithelium as illustrated by a rapid change in Isc following stimulation (+14, +7, -28 µA/cm(2) for glucose, glutamine and Intralipid, respectively, in the jejunum; +132, +42, -14, -56 µA/cm(2) for glucose, glutamine, fructose and Intralipid in the ileum). Addition of 5% Intralipid to the luminal side increased TER in both jejunum (+10 Ω/cm(2)) and Ileum (+10 Ω/cm(2)). Glutamine triggered a significant (p

BACKGROUND: The barrier function of the intestinal epithelium is a first-line of defense that is compromised in IBD. We have shown that epithelia treated with dinitrophenol (DNP: uncouples oxidative phosphorylation) have a dramatic impairment in their barrier function, and internalize non-invasive commensal E. coli. Mitochondria do not function in isolation; they are closely aligned with the endoplasmic reticulum (ER). AIMS: To investigate the interaction of mitochondrial dysfunction, ER stress and autophagic processes as a key determinant of epithelial-bacterial interaction, transcellular permeability and the fate of commensal bacteria that gain access to the intracellular compartment as a consequence of metabolic stress. METHODS: METHODS: Human colonic biopsies were mounted in Ussing chambers, murine epithelial colonic organoids were re-suspended and grown as a monolayer, and human colon-derived epithelial cell lines were cultured on transwell filters or on plastic. Bacteria were added to the luminal buffer and the tissue treated with DNP ± the ER stressor, tunicamycin (TM), and bacterial internalization and translocation assessed. Mechanistic studies involved measuring ATP production, immunoblotting, assessment of LC3 activation for autophagy and gene knock-down (KD) by siRNA and CRISPR/cas9. RESULTS: RESULTS: DNP promoted the translocation and internalization of E. coli and, remarkably, TM reduced this barrier defect. TM did not prevent the DNP-evoked drop in ATP or the rate of epithelial update of inert beads, but E. coli, DNP and TM-treated cells had increased autophagy. The effect of TM was lost in cells lacking the autophagy protein, ATG16L1, suggesting that the ER-stress promoted killing of the internalized bacteria. Of the 3 major arms of the ER stress response, only KD of ATF6 ablated the TM antagonism of the DNP-evoked barrier effect; the increase in autophagy evoked by TM was absent in ATF6 KD epithelia. Pharmacological studies indicate that the kinase, DAPK1 is required from the ER stress-ATF6 signal to drive autophagy to kill the internalized E. coli. CONCLUSIONS: Conclusion: The ATF6 arm of ER stress antagonizes metabolic stress-induced decreases in epithelial barrier function to commensal bacteria by promoting xenophagy via upregulation of DAPK1. FUNDING AGENCIES: CAG, CIHRAIHS, HPI-NSERC, CNPq

NOT PUBLISHED AT AUTHOR’S REQUEST Funding Agencies: CIHRAlberta Innovates, Human Frontier Science Program

BACKGROUND: The inflammatory microenvironment in the gut contains a variety of proteases which are known to be increased in patient samples in both IBD and CRC. How these proteases and their proteolytic peptide products contribute to wound resolution has been poorly elucidated. A cellular switch from a “barrier” to a “migration/repair” phenotype is required for healing in IBD, but is also a hallmark of tumorigenesis. Proteolytic degradation of epithelial E-cadherin (Ecad) is necessary for this process, we have been studying the biological activity small peptide fragments of Ecad generated by neutrophil elastase, an inflammatory protease elevated in IBD. We have been studying the ability of proteases to induce a switch in the colonic epithelium from a barrier to a repair phenotype (epithelial to mesenchymal transition [EMT]) characterized by increased migration and disrupted homeostasis. AIMS: To test the hypothesis that inflammatory proteases process Ecad into small bioactive peptides that contribute to wound resolution. METHODS: Recombinant Ecad was incubated with neutrophil elastase (NE) in vitro to produce NE-dependent Ecad peptides. Six of these peptides shared partial homology with Ecad peptides identified by mass spectrometry in IBD patient samples, and were chosen for further characterization. Peptides were synthesized and assayed for their ability to alter wound healing capacity, proliferation, cell spreading, and cytotoxicity in Caco-2 cells using the IncuCyte™ live-cell imaging system for 48 hours following exposure to 1, 10, and 100 mg/mL concentrations of peptides. All analysis was done using the IncuCyte™ ZOOM platform in conjunction with ImageJ software. RESULTS: We have identified 6 Ecad peptides produced by neutrophil elastase activity that appear to be increased in IBD patient tissue. We have characterized these peptides to have novel biological roles in altering wound resolution and proliferation rates, with 3 peptides showing significantly increased wound healing capacity at at least one concentration. These 3 peptides (KAADTDPTAPPYD, NRNTGVISVV, and LPPEDDTRDNV) showed improved wound closures of approximately 7–10% under serum free conditions and 5–10% under full serum conditions compared to controls. Preliminary data also suggests that these peptides appear to have a positive effect on proliferation rates, and appear to alter cellular morphology of cells to that of a more flattened cell type, typical of repair programming of cells to cover a denuded area. CONCLUSIONS: Our data reveal a novel role for proteolytic processing of Ecad under inflammatory conditions, producing bioactive peptides that drive a wound-healing phenotype in epithelial cells in response to damage. FUNDING AGENCIES: CIHRUniversity of Calgary Faculty Seed Grants