Publications by authors named "Wallace K MacNaughton"

54 Publications

Intestinal fungi are causally implicated in microbiome assembly and immune development in mice.

Nat Commun 2020 05 22;11(1):2577. Epub 2020 May 22.

Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada.

The gut microbiome consists of a multi-kingdom microbial community. Whilst the role of bacteria as causal contributors governing host physiological development is well established, the role of fungi remains to be determined. Here, we use germ-free mice colonized with defined species of bacteria, fungi, or both to differentiate the causal role of fungi on microbiome assembly, immune development, susceptibility to colitis, and airway inflammation. Fungal colonization promotes major shifts in bacterial microbiome ecology, and has an independent effect on innate and adaptive immune development in young mice. While exclusive fungal colonization is insufficient to elicit overt dextran sulfate sodium-induced colitis, bacterial and fungal co-colonization increase colonic inflammation. Ovalbumin-induced airway inflammation reveals that bacterial, but not fungal colonization is necessary to decrease airway inflammation, yet fungi selectively promotes macrophage infiltration in the airway. Together, our findings demonstrate a causal role for fungi in microbial ecology and host immune functionality, and therefore prompt the inclusion of fungi in therapeutic approaches aimed at modulating early life microbiomes.
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http://dx.doi.org/10.1038/s41467-020-16431-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244730PMC
May 2020

Acute regulation of intestinal ion transport and permeability in response to luminal nutrients: the role of the enteric nervous system.

Am J Physiol Gastrointest Liver Physiol 2020 02 11;318(2):G254-G264. Epub 2019 Nov 11.

Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada.

The small intestine regulates barrier function to absorb nutrients while avoiding the entry of potentially harmful substances or bacteria. Barrier function is dynamically regulated in part by the enteric nervous system (ENS). The role of the ENS in regulating barrier function in response to luminal nutrients is not well understood. We hypothesize that the ENS regulates intestinal permeability and ion flux in the small intestine in response to luminal nutrients. Segments of jejunum and ileum from mice were mounted in Ussing chambers. Transepithelial electrical resistance (TER), short-circuit current (), and permeability to 4-kDa FITC-dextran (FD4) were recorded after mucosal stimulation with either glucose, fructose, glutamine (10 mM), or 5% Intralipid. Mucosal lipopolysaccharide (1 mg/mL) was also studied. Enteric neurons were inhibited with tetrodotoxin (TTX; 0.5 μM) or activated with veratridine (10 μM). Enteric glia were inhibited with the connexin-43 blocker Gap26 (20 μM). Glucose, glutamine, Intralipid, and veratridine acutely modified in the jejunum and ileum, but the effect of nutrients on was insensitive to TTX. TTX, Gap26, and veratridine treatment did not affect baseline TER or permeability. Intralipid acutely decreased permeability to FD4, while LPS increased it. TTX pretreatment abolished the effect of Intralipid and exacerbated the LPS-induced increase in permeability. Luminal nutrients and enteric nerve activity both affect ion flux in the mouse small intestine acutely but independently of each other. Neither neuronal nor glial activity is required for the maintenance of baseline intestinal permeability; however, neuronal activity is essential for the acute regulation of intestinal permeability in response to luminal lipids and lipopolysaccharide. Luminal nutrients and enteric nerve activity both affect ion transport in the mouse small intestine acutely, but independently of each other. Activation or inhibition of the enteric neurons does not affect intestinal permeability, but enteric neural activity is essential for the acute regulation of intestinal permeability in response to luminal lipids and lipopolysaccharide. The enteric nervous system regulates epithelial homeostasis in the small intestine in a time-dependent, region- and stimulus-specific manner.
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http://dx.doi.org/10.1152/ajpgi.00186.2019DOI Listing
February 2020

N-Terminomics/TAILS Profiling of Proteases and Their Substrates in Ulcerative Colitis.

ACS Chem Biol 2019 11 21;14(11):2471-2483. Epub 2019 Aug 21.

Department of Physiology and Pharmacology , University of Calgary , Calgary , Alberta , Canada T2N 4N1.

Dysregulated protease activity is often implicated in the initiation of inflammation and immune cell recruitment in gastrointestinal inflammatory diseases. Using N-terminomics/TAILS (terminal amine isotopic labeling of substrates), we compared proteases, along with their substrates and inhibitors, between colonic mucosal biopsies of healthy patients and those with ulcerative colitis (UC). Among the 1642 N-termini enriched using TAILS, increased endogenous processing of proteins was identified in UC compared to healthy patients. Changes in the reactome pathways for proteins associated with metabolism, adherens junction proteins (E-cadherin, liver-intestinal cadherin, catenin alpha-1, and catenin delta-1), and neutrophil degranulation were identified between the two groups. Increased neutrophil infiltration and distinct proteases observed in ulcerative colitis may result in extensive break down, altered processing, or increased remodeling of adherens junctions and other cellular functions. Analysis of the preferred proteolytic cleavage sites indicated that the majority of proteolytic activity and processing comes from host proteases, but that key microbial proteases may also play a role in maintaining homeostasis. Thus, the identification of distinct proteases and processing of their substrates improves the understanding of dysregulated proteolysis in normal intestinal physiology and ulcerative colitis.
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http://dx.doi.org/10.1021/acschembio.9b00608DOI Listing
November 2019

N-Terminomics/TAILS Profiling of Proteases and Their Substrates in Ulcerative Colitis.

ACS Chem Biol 2019 11 21;14(11):2471-2483. Epub 2019 Aug 21.

Department of Physiology and Pharmacology , University of Calgary , Calgary , Alberta , Canada T2N 4N1.

Dysregulated protease activity is often implicated in the initiation of inflammation and immune cell recruitment in gastrointestinal inflammatory diseases. Using N-terminomics/TAILS (terminal amine isotopic labeling of substrates), we compared proteases, along with their substrates and inhibitors, between colonic mucosal biopsies of healthy patients and those with ulcerative colitis (UC). Among the 1642 N-termini enriched using TAILS, increased endogenous processing of proteins was identified in UC compared to healthy patients. Changes in the reactome pathways for proteins associated with metabolism, adherens junction proteins (E-cadherin, liver-intestinal cadherin, catenin alpha-1, and catenin delta-1), and neutrophil degranulation were identified between the two groups. Increased neutrophil infiltration and distinct proteases observed in ulcerative colitis may result in extensive break down, altered processing, or increased remodeling of adherens junctions and other cellular functions. Analysis of the preferred proteolytic cleavage sites indicated that the majority of proteolytic activity and processing comes from host proteases, but that key microbial proteases may also play a role in maintaining homeostasis. Thus, the identification of distinct proteases and processing of their substrates improves the understanding of dysregulated proteolysis in normal intestinal physiology and ulcerative colitis.
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http://dx.doi.org/10.1021/acschembio.9b00608DOI Listing
November 2019

Proteolytic Processing of the Epithelial Adherens Junction Molecule E-Cadherin by Neutrophil Elastase Generates Short Peptides With Novel Wound-Healing Bioactivity.

Cell Mol Gastroenterol Hepatol 2019 4;7(2):483-486.e8. Epub 2018 Nov 4.

Department of Physiology and Pharmacology, Gastrointestinal Research Group, University of Calgary, Calgary, Alberta, Canada. Electronic address:

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http://dx.doi.org/10.1016/j.jcmgh.2018.10.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410347PMC
May 2019

Itch induced by peripheral mu opioid receptors is dependent on TRPV1-expressing neurons and alleviated by channel activation.

Sci Rep 2018 10 19;8(1):15551. Epub 2018 Oct 19.

Inflammation Research Network-Snyder Institute for Chronic Diseases, and Alberta Children's Hospital Research Institute, Department of Physiology & Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.

Opioids remain the gold standard for the treatment of moderate to severe pain. However, their analgesic properties come with important side effects, including pruritus, which occurs frequently after systemic or neuraxial administration. Although part of the opioid-induced itch is mediated centrally, recent evidence shows that the opioid receptor system in the skin also modulates itch. The goal of our study was to identify the peripherally located transducer mechanisms involved in opioid-induced pruritus. Scratching behaviors in response to an intradermal injection of the mu-opioid receptor (MOR) agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) was quantified in mast cell-, PAR2- and TRPV1-deficient mice or following ablation of TRPV1+ sensory neurons. We found that mast cells-/-, PAR-2-/-, or TRPV1-/- mice still exhibit DAMGO-induced itch responses. However, we show that ablation of TRPV1+ neurons or acute TRPV1 activation by capsaicin abolishes DAMGO-induced itch. Overall, our work shows that peripheral DAMGO-induced itch is dependent on the presence of TRPV1-expressing pruriceptors, but not the TRPV1 channel itself. Activation of these fibers by capsaicin prevents the opioid-induced itch.
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http://dx.doi.org/10.1038/s41598-018-33620-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195532PMC
October 2018

Inhibition of Intestinal Epithelial Wound Healing through Protease-Activated Receptor-2 Activation in Caco2 Cells.

J Pharmacol Exp Ther 2018 11 6;367(2):382-392. Epub 2018 Sep 6.

Departments of Physiology and Pharmacology (E.H.F., M.H.G., W.K.M.) and Medicine (P.L.B.), Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada

The mechanisms of epithelial wound healing are not completely understood, especially in the context of proteases and their receptors. It was recently shown that activation of protease-activated receptor-2 (PAR2) on intestinal epithelial cells induced the expression of cyclooxygenase-2 (COX-2), which has protective functions in the gastrointestinal tract. It was hypothesized that PAR2-induced COX-2 could enhance wound healing in intestinal epithelial cells. Caco2 cells were used to model epithelial wound healing of circular wounds. Cellular proliferation was studied with a 5-ethynyl-2'-deoxyuridine assay, and migration was studied during wound healing in the absence of proliferation. Immunofluorescence was used to visualize E-cadherin and F-actin, and the cellular transcription profile during wound healing and PAR2 activation was explored with RNA sequencing. PAR2 activation inhibited Caco2 wound healing by reducing cell migration, independently of COX-2 activity. Interestingly, even though migration was reduced, proliferation was increased. When the actin dynamics and cell-cell junctions were investigated, PAR2 activation was found to induce actin cabling and prevent the internalization of E-cadherin. To further investigate the effect of PAR2 on transcriptionally dependent wound healing, RNA sequencing was performed. This analysis revealed that PAR2 activation, in the absence of wounding, induced a similar transcriptional profile compared with wounding alone. These findings represent a novel effect of PAR2 activation on the mechanisms of epithelial cell wound healing that could influence the resolution of intestinal inflammation.
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http://dx.doi.org/10.1124/jpet.118.249524DOI Listing
November 2018

Interferon gamma decreases intestinal epithelial aquaporin 3 expression through downregulation of constitutive transcription.

J Mol Med (Berl) 2018 10 8;96(10):1081-1093. Epub 2018 Aug 8.

Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada.

Aquaporin (AQP) 3 expression is altered in inflammatory bowel diseases, although the exact mechanisms regulating AQP abundance are unclear. Although interferon gamma (IFNγ) is centrally involved in intestinal inflammation, the effect of this cytokine on AQP3 expression remains unknown. HT-29 human colonic epithelial cells were treated with IFNγ to assess AQP3 mRNA expression by real-time RT-PCR and functional protein expression through the uptake of radiolabelled glycerol. Transient knockdown of signal transducer and activator of transcription 1 (STAT1), STAT3, Sp1, and Sp3 were performed to determine the involvement of these transcription factors in the IFNγ-induced signalling cascade. AQP3 promoter regions involved in the response to IFNγ were assessed using a luciferase reporter system. Likewise, enteroids derived from human colonic biopsies were also treated with IFNγ to assess for changes in AQP3 mRNA expression. IFNγ decreased AQP3 mRNA expression in HT-29 cells in a time- and concentration-dependent manner and reduced functional AQP3 protein expression (decreased H-labelled glycerol uptake). IFNγ also reduced AQP3 expression in enteroids derived from human colonic biopsies. Knockdown of STAT1 partially prevented the IFNγ-induced downregulation of AQP3 expression, whereas STAT3 and Sp3 knockdowns resulted in increased baseline expression of AQP3 but did not alter IFNγ-induced downregulation. Constitutive transcription of AQP3 is downregulated by IFNγ as demonstrated using the luciferase reporter system, with Sp3 bound to the AQP3 promoter as shown by chromatin immunoprecipitation. AQP3 constitutive transcription in intestinal epithelial cells is downregulated by IFNγ. This response requires STAT1 that is postulated to drive the downregulation of AQP3 expression through increased acetylation or decreased deacetylation the AQP3 promoter, ultimately resulting in decreased constitutive transcription of AQP3.

Key Messages: • IFNγ suppresses the expression of AQP3 in intestinal epithelial cells. • Proximal AQP3 promoter elements are sufficient to drive constitutive expression and mediate the IFNγ-induced downregulation of AQP3 mRNA expression. • IFNγ-induced suppression of AQP3 is dependent upon STAT1 expression, but not STAT3, Sp1, or Sp3.
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http://dx.doi.org/10.1007/s00109-018-1681-2DOI Listing
October 2018

ER-stress mobilization of death-associated protein kinase-1-dependent xenophagy counteracts mitochondria stress-induced epithelial barrier dysfunction.

J Biol Chem 2018 03 9;293(9):3073-3087. Epub 2018 Jan 9.

From the Gastrointestinal Research Group, Departments of Physiology and Pharmacology and

The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD, and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here, we treated human colonic biopsies, epithelial colonoids, and epithelial cells with an uncoupler of oxidative phosphorylation, dinitrophenol (DNP), with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6, an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis, induction of ER stress ( the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather, specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note, epithelia with a Crohn's disease-susceptibility mutation in the autophagy gene exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress-ATF6-DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria, thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation.
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http://dx.doi.org/10.1074/jbc.RA117.000809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836131PMC
March 2018

Tumor necrosis factor decreases aquaporin 3 expression in intestinal epithelial cells through inhibition of constitutive transcription.

Physiol Rep 2017 Oct 16;5(19). Epub 2017 Oct 16.

Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta, Canada

Inflammatory diseases of the gut are associated with altered electrolyte and water transport, leading to the development of diarrhea. Epithelially expressed aquaporins (AQPs) are downregulated in inflammation, although the mechanisms involved are not known. We hypothesized that AQP3 expression in intestinal epithelial cells is altered in intestinal inflammation and that these changes are driven by tumor necrosis factor (TNF) Human colonic adenocarcinoma (HT-29) cells were treated with TNF to investigate signaling mechanisms in vitro. AQP3 expression was assessed by real-time PCR and radiolabeled glycerol uptake, with select inhibitors and a luciferase reporter construct used to further elucidate intracellular signaling. AQP3 expression was downregulated in HT-29 cells treated with TNF Luciferase reporter construct experiments revealed that TNF downregulated constitutive transcriptional activity of the AQP3 promoter, and inhibition of MEK/ERK and nuclear factor B (NF-B) signaling prevented the decrease in AQP3 mRNA expression. Constitutive AQP3 expression was suppressed by specificity protein (Sp) 3, and knockdown of this transcription factor bound to the AQP3 promoter was able to partially prevent the TNF-induced downregulation of AQP3. TNF signals through MEK/ERK and NF-B to enhance the negative transcriptional control of AQP3 expression exerted by Sp3. Similar mechanisms regulate numerous ion channels, suggesting a common mechanism by which both ion and water transport are altered in inflammation.
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http://dx.doi.org/10.14814/phy2.13451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5641937PMC
October 2017

A simple, cost-effective method for generating murine colonic 3D enteroids and 2D monolayers for studies of primary epithelial cell function.

Am J Physiol Gastrointest Liver Physiol 2017 Nov 27;313(5):G467-G475. Epub 2017 Jul 27.

Department of Physiology and Pharmacology and Gastrointestinal Research Group, University of Calgary, Calgary, Alberta, Canada;

Cancer cell lines have been the mainstay of intestinal epithelial experimentation for decades, due primarily to their immortality and ease of culture. However, because of the inherent biological abnormalities of cancer cell lines, many cellular biologists are currently transitioning away from these models and toward more representative primary cells. This has been particularly challenging, but recent advances in the generation of intestinal organoids have brought the routine use of primary cells within reach of most epithelial biologists. Nevertheless, even with the proliferation of publications that use primary intestinal epithelial cells, there is still a considerable amount of trial and error required for laboratories to establish a consistent and reliable method to culture three-dimensional (3D) intestinal organoids and primary epithelial monolayers. We aim to minimize the time other laboratories spend troubleshooting the technique and present a standard method for culturing primary epithelial cells. Therefore, we have described our optimized, high-yield, cost-effective protocol to grow 3D murine colonoids for more than 20 passages and our detailed methods to culture these cells as confluent monolayers for at least 14 days, enabling a wide variety of potential future experiments. By supporting and expanding on the current literature of primary epithelial culture optimization and detailed use in experiments, we hope to help enable the widespread adoption of these innovative methods and allow consistency of results obtained across laboratories and institutions. Primary intestinal epithelial monolayers are notoriously difficult to maintain culture, even with the recent advances in the field. We describe, in detail, the protocols required to maintain three-dimensional cultures of murine colonoids and passage these primary epithelial cells to confluent monolayers in a standardized, high-yield and cost-effective manner.
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http://dx.doi.org/10.1152/ajpgi.00152.2017DOI Listing
November 2017

Signaling pathways induced by serine proteases to increase intestinal epithelial barrier function.

PLoS One 2017 3;12(7):e0180259. Epub 2017 Jul 3.

Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta, Canada.

Changes in barrier function of the gastrointestinal tract are thought to contribute to the inflammatory bowel diseases Crohn's disease and ulcerative colitis. Previous work in our lab demonstrated that apical exposure of intestinal epithelial cell lines to serine proteases results in an increase in transepithelial electrical resistance (TER). However, the underlying mechanisms governing this response are unclear. We aimed to determine the requirement for proteolytic activity, epidermal growth factor receptor (EGFR) activation, and downstream intracellular signaling in initiating and maintaining enhanced barrier function following protease treatment using a canine intestinal epithelial cell line (SCBN). We also examined the role of phosphorylation of myosin regulatory light chain on the serine protease-induced increase in TER through. It was found that proteolytic activity of the serine proteases trypsin and matriptase is required to initiate and maintain the protease-mediated increase in TER. We also show that MMP-independent EGFR activation is essential to the sustained phase of the protease response, and that Src kinases may mediate EGFR transactivation. PI3-K and ERK1/2 signaling were important in reaching a maximal increase in TER following protease stimulation; however, their upstream activators are yet to be determined. CK2 inhibition prevented the increase in TER induced by serine proteases. The bradykinin B(2) receptor was not involved in the change in TER in response to serine proteases, and no change in phosphorylation of MLC was observed after trypsin or matriptase treatment. Taken together, our data show a requirement for ongoing proteolytic activity, EGFR transactivation, as well as downstream PI3-K, ERK1/2, and CK2 signaling in protease-mediated barrier enhancement of intestinal epithelial cells. The pathways mediating enhanced barrier function by proteases may be novel therapeutic targets for intestinal disorders characterized by disrupted epithelial barrier function.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180259PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495298PMC
October 2017

Hydrogen Sulfide-Releasing Therapeutics: Translation to the Clinic.

Antioxid Redox Signal 2018 06 15;28(16):1533-1540. Epub 2017 May 15.

3 Universidade Camilo Castelo Branco , Department of Medicine, Fernandopolis, Brazil .

Significance: Shortly after the discovery of the role of hydrogen sulfide (HS) in many physiological and pathological processes, attempts were made to develop novel pharmaceuticals that may be of benefit for treatment or prevention of a wide range of disorders. The promise of HS-based therapeutics is now being demonstrated in clinical trials. Recent Advances: HS-releasing drugs, such as SG1002 for cardiovascular disorders, and ATB-346 for arthritis, have progressed into clinical trials and have shown considerable promise. Some older drugs, such as zofenopril, have now been recognized to produce at least some of the beneficial effects through release of HS.

Critical Issues: There remains a need to better understand the underlying mechanisms for some of the observed effects of HS-releasing drugs in a clinical setting, such as the marked increase in analgesic potency that has been observed with ATB-346.

Future Directions: The proof-of-concept clinical studies reviewed herein pave the way for examination, in a clinical setting, of several other potential applications of HS-based drugs in a wide range of disorders, including diabetes, hypertension, and cancer chemoprevention. Antioxid. Redox Signal. 28, 1533-1540.
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http://dx.doi.org/10.1089/ars.2017.7068DOI Listing
June 2018

Constipation-Predominant Irritable Bowel Syndrome Females Have Normal Colonic Barrier and Secretory Function.

Am J Gastroenterol 2017 Jun 21;112(6):913-923. Epub 2017 Mar 21.

Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota, USA.

Objectives: The objective of this study was to determine whether constipation-predominant irritable bowel syndrome (IBS-C) is associated with changes in intestinal barrier and secretory function.

Methods: A total of 19 IBS-C patients and 18 healthy volunteers (all females) underwent saccharide excretion assay (0.1 g C mannitol and 1 g lactulose), measurements of duodenal and colonic mucosal barrier (transmucosal resistance (TMR), macromolecular and Escherichia coli Bio-Particle translocation), mucosal secretion (basal and acetylcholine (Ach)-evoked short-circuit current (Isc)), in vivo duodenal mucosal impedance, circulating endotoxins, and colonic tight junction gene expression.

Results: There were no differences in the in vivo measurements of barrier function between IBS-C patients and healthy controls: cumulative excretion of C mannitol (0-2 h mean (s.e.m.); IBS-C: 12.1 (0.9) mg vs. healthy: 13.2 (0.8) mg) and lactulose (8-24 h; IBS-C: 0.9 (0.5) mg vs. healthy: 0.5 (0.2) mg); duodenal impedance IBS-C: 729 (65) Ω vs. healthy: 706 (43) Ω; plasma mean endotoxin activity level IBS-C: 0.36 (0.03) vs. healthy: 0.35 (0.02); and in colonic mRNA expression of occludin, zonula occludens (ZO) 1-3, and claudins 1-12 and 14-19. The ex vivo findings were consistent, with no group differences: duodenal TMR (IBS-C: 28.2 (1.9) Ω cm vs. healthy: 29.8 (1.9) Ω cm) and colonic TMR (IBS-C: 19.1 (1.1) Ω cm vs. healthy: 17.6 (1.7) Ω cm); fluorescein isothiocyanate (FITC)-dextran (4 kDa) and E. coli Bio-Particle flux. Colonic basal Isc was similar, but duodenal basal Isc was lower in IBS-C (43.5 (4.5) μA cm) vs. healthy (56.9 (4.9) μA cm), P=0.05. Ach-evoked ΔIsc was similar.

Conclusions: Females with IBS-C have normal colonic barrier and secretory function. Basal duodenal secretion is decreased in IBS-C.
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http://dx.doi.org/10.1038/ajg.2017.48DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5502210PMC
June 2017

The serine protease-mediated increase in intestinal epithelial barrier function is dependent on occludin and requires an intact tight junction.

Am J Physiol Gastrointest Liver Physiol 2016 09 4;311(3):G466-79. Epub 2016 Aug 4.

Department of Physiology and Pharmacology and Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; and

Barrier dysfunction is a characteristic of the inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis. Understanding how the tight junction is modified to maintain barrier function may provide avenues for treatment of IBD. We have previously shown that the apical addition of serine proteases to intestinal epithelial cell lines causes a rapid and sustained increase in transepithelial electrical resistance (TER), but the mechanisms are unknown. We hypothesized that serine proteases increase barrier function through trafficking and insertion of tight junction proteins into the membrane, and this could enhance recovery of a disrupted monolayer after calcium switch or cytokine treatment. In the canine epithelial cell line, SCBN, we showed that matriptase, an endogenous serine protease, could potently increase TER. Using detergent solubility-based cell fractionation, we found that neither trypsin nor matriptase treatment changed levels of tight junction proteins at the membrane. In a fast calcium switch assay, serine proteases did not enhance the rate of recovery of the junction. In addition, serine proteases could not reverse barrier disruption induced by IFNγ and TNFα. We knocked down occludin in our cells using siRNA and found this prevented the serine protease-induced increase in TER. Using fluorescence recovery after photobleaching (FRAP), we found serine proteases induce a greater mobile fraction of occludin in the membrane. These data suggest that a functional tight junction is needed for serine proteases to have an effect on TER, and that occludin is a crucial tight junction protein in this mechanism.
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http://dx.doi.org/10.1152/ajpgi.00441.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5076006PMC
September 2016

Protective Actions of Epithelial 5-Hydroxytryptamine 4 Receptors in Normal and Inflamed Colon.

Gastroenterology 2016 11 29;151(5):933-944.e3. Epub 2016 Jul 29.

Neurological Sciences, University of Vermont, Burlington, Vermont. Electronic address:

Background & Aims: The 5-hydroxytryptamine receptor 4 (5-HTR or HTR) is expressed in the colonic epithelium but little is known about its functions there. We examined whether activation of colonic epithelial 5-HTR protects colons of mice from inflammation.

Methods: The 5-HTR agonist tegaserod (1 mg/kg), the 5-HTR antagonist GR113808 (1 mg/kg), or vehicle (control) were delivered by enema to wild-type or 5-HTR knockout mice at the onset of, or during, active colitis, induced by administration of dextran sodium sulfate or trinitrobenzene sulfonic acid. Inflammation was measured using the colitis disease activity index and by histologic analysis of intestinal tissues. Epithelial proliferation, wound healing, and resistance to oxidative stress-induced apoptosis were assessed, as was colonic motility.

Results: Rectal administration of tegaserod reduced the severity of colitis compared with mice given vehicle, and accelerated recovery from active colitis. Rectal tegaserod did not improve colitis in 5-HTR knockout mice, and intraperitoneally administered tegaserod did not protect wild-type mice from colitis. Tegaserod increased proliferation of crypt epithelial cells. Stimulation of 5-HTR increased Caco-2 cell migration and reduced oxidative stress-induced apoptosis; these actions were blocked by co-administration of the 5-HTR antagonist GR113808. In noninflamed colons of wild-type mice not receiving tegaserod, inhibition of 5-HTRs resulted in signs of colitis within 3 days. In these mice, epithelial proliferation decreased and bacterial translocation to the liver and spleen was detected. Daily administration of tegaserod increased motility in inflamed colons of guinea pigs and mice, whereas administration of GR113808 disrupted motility in animals without colitis.

Conclusions: 5-HTR activation maintains motility in healthy colons of mice and guinea pigs, and reduces inflammation in colons of mice with colitis. Agonists might be developed as treatments for patients with inflammatory bowel diseases.
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http://dx.doi.org/10.1053/j.gastro.2016.07.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5159265PMC
November 2016

Probiotics Improve Inflammation-Associated Sickness Behavior by Altering Communication between the Peripheral Immune System and the Brain.

J Neurosci 2015 Jul;35(30):10821-30

Immunology Research Group and

Unlabelled: Patients with systemic inflammatory diseases (e.g., rheumatoid arthritis, inflammatory bowel disease, chronic liver disease) commonly develop debilitating symptoms (i.e., sickness behaviors) that arise from changes in brain function. The microbiota-gut-brain axis alters brain function and probiotic ingestion can influence behavior. However, how probiotics do this remains unclear. We have previously described a novel periphery-to-brain communication pathway in the setting of peripheral organ inflammation whereby monocytes are recruited to the brain in response to systemic TNF-α signaling, leading to microglial activation and subsequently driving sickness behavior development. Therefore, we investigated whether probiotic ingestion (i.e., probiotic mixture VSL#3) alters this periphery-to-brain communication pathway, thereby reducing subsequent sickness behavior development. Using a well characterized mouse model of liver inflammation, we now show that probiotic (VSL#3) treatment attenuates sickness behavior development in mice with liver inflammation without affecting disease severity, gut microbiota composition, or gut permeability. Attenuation of sickness behavior development was associated with reductions in microglial activation and cerebral monocyte infiltration. These events were paralleled by changes in markers of systemic immune activation, including decreased circulating TNF-α levels. Our observations highlight a novel pathway through which probiotics mediate cerebral changes and alter behavior. These findings allow for the potential development of novel therapeutic interventions targeted at the gut microbiome to treat inflammation-associated sickness behaviors in patients with systemic inflammatory diseases.

Significance Statement: This research shows that probiotics, when eaten, can improve the abnormal behaviors (including social withdrawal and immobility) that are commonly associated with inflammation. Probiotics are able to cause this effect within the body by changing how the immune system signals the brain to alter brain function. These findings broaden our understanding of how probiotics may beneficially affect brain function in the context of inflammation occurring within the body and may open potential new therapeutic alternatives for the treatment of these alterations in behavior that can greatly affect patient quality of life.
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http://dx.doi.org/10.1523/JNEUROSCI.0575-15.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605112PMC
July 2015

Proteases and their receptors as mediators of inflammation-associated colon cancer.

Curr Pharm Des 2015 ;21(21):2983-92

Department of Physiology and Pharmacology, University of Calgary, 3330 Hospital Dr NW, Calgary, AB, T2N 4N1 Canada.

Chronic inflammation increases the risk of developing cancer. For example, patients with severe and prolonged inflammatory bowel disease, particularly ulcerative colitis, have a significantly higher risk of developing colorectal cancer. Serine proteases coordinating the coagulation cascade and immune cell proteases play important roles in regulating the inflammatory response through their actions on protease-activated receptors (PAR). PARs and their activating proteases have also been implicated in many cancers, including CRC. Importantly, the actions of proteases could be important for mediating the transition from chronic inflammation to cancer. PAR activation has been shown to have pro-tumourigenic effects including the production of matrix metalloproteinases that can promote tumour cell growth and metastasis, and transactivation of the epidermal growth factor receptor, which is a main target for cancer treatment. Additionally, PAR activation can also result in increased expression of cyclooxygenase (COX)-2, an important enzyme mediating inflammation, resolution, and cancer progression. In this review, we will highlight our current knowledge about the effects of proteases and their receptors on intestinal inflammation and cancer, and explore the potential role of PAR-induced COX-2 on colitis-associated cancer.
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http://dx.doi.org/10.2174/1381612821666150514104800DOI Listing
March 2016

Inhibiting Inducible Nitric Oxide Synthase in Enteric Glia Restores Electrogenic Ion Transport in Mice With Colitis.

Gastroenterology 2015 Aug 9;149(2):445-55.e3. Epub 2015 Apr 9.

Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada. Electronic address:

Background & Aims: Disturbances in the control of ion transport lead to epithelial barrier dysfunction in patients with colitis. Enteric glia regulate intestinal barrier function and colonic ion transport. However, it is not clear whether enteric glia are involved in epithelial hyporesponsiveness. We investigated enteric glial regulation of ion transport in mice with trinitrobenzene sulfonic acid- or dextran sodium sulfate-induced colitis and in Il10(-/-) mice.

Methods: Electrically evoked ion transport was measured in full-thickness segments of colon from CD1 and Il10(-/-) mice with or without colitis in Ussing chambers. Nitric oxide (NO) production was assessed using amperometry. Bacterial translocation was investigated in the liver, spleen, and blood of mice.

Results: Electrical stimulation of the colon evoked a tetrodotoxin-sensitive chloride secretion. In mice with colitis, ion transport almost completely disappeared. Inhibiting inducible NO synthase (NOS2), but not neuronal NOS (NOS1), partially restored the evoked secretory response. Blocking glial function with fluoroacetate, which is not a NOS2 inhibitor, also partially restored ion transport. Combined NOS2 inhibition and fluoroacetate administration fully restored secretion. Epithelial responsiveness to vasoactive intestinal peptide was increased after enteric glial function was blocked in mice with colitis. In colons of mice without colitis, NO was produced in the myenteric plexus almost completely via NOS1. NO production was increased in mice with colitis, compared with mice without colitis; a substantial proportion of NOS2 was blocked by fluoroacetate administration. Inhibition of enteric glial function in vivo reduced the severity of trinitrobenzene sulfonic acid-induced colitis and associated bacterial translocation.

Conclusions: Increased production of NOS2 in enteric glia contributes to the dysregulation of intestinal ion transport in mice with colitis. Blocking enteric glial function in these mice restores epithelial barrier function and reduces bacterial translocation.
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http://dx.doi.org/10.1053/j.gastro.2015.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4516675PMC
August 2015

Interferon-γ suppresses intestinal epithelial aquaporin-1 expression via Janus kinase and STAT3 activation.

PLoS One 2015 20;10(3):e0118713. Epub 2015 Mar 20.

Inflammation Research Network and Department of Physiology and Pharmacology, University of Calgary, Calgary, Canada.

Inflammatory bowel diseases are associated with dysregulated electrolyte and water transport and resultant diarrhea. Aquaporins are transmembrane proteins that function as water channels in intestinal epithelial cells. We investigated the effect of the inflammatory cytokine, interferon-γ, which is a major player in inflammatory bowel diseases, on aquaporin-1 expression in a mouse colonic epithelial cell line, CMT93. CMT93 monolayers were exposed to 10 ng/mL interferon-γ and aquaporin-1 mRNA and protein expressions were measured by real-time PCR and western blot, respectively. In other experiments, CMT93 cells were pretreated with inhibitors or were transfected with siRNA to block the effects of Janus kinases, STATs 1 and 3, or interferon regulatory factor 2, prior to treatment with interferon-γ. Interferon-γ decreased aquaporin-1 expression in mouse intestinal epithelial cells in a manner that did not depend on the classical STAT1/JAK2/IRF-1 pathway, but rather, on an alternate Janus kinase (likely JAK1) as well as on STAT3. The pro-inflammatory cytokine, interferon-γ may contribute to diarrhea associated with intestinal inflammation in part through regulation of the epithelial aquaporin-1 water channel via a non-classical JAK/STAT receptor signalling pathway.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0118713PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405000PMC
December 2015

Proteinase-activated receptor 2 (PAR2) decreases apoptosis in colonic epithelial cells.

J Biol Chem 2014 Dec 20;289(49):34366-77. Epub 2014 Oct 20.

From the Department of Physiology and Pharmacology, the Inflammation Research Network, and the Snyder Institute for Chronic Diseases, University of Calgary, Alberta T2N 4N1, Canada

Mucosal biopsies from inflamed colon of inflammatory bowel disease patients exhibit elevated epithelial apoptosis compared with those from healthy individuals, disrupting mucosal homeostasis and perpetuating disease. Therapies that decrease intestinal epithelial apoptosis may, therefore, ameliorate inflammatory bowel disease, but treatments that specifically target apoptotic pathways are lacking. Proteinase-activated receptor-2 (PAR2), a G protein-coupled receptor activated by trypsin-like serine proteinases, is expressed on intestinal epithelial cells and stimulates mitogenic pathways upon activation. We sought to determine whether PAR2 activation and signaling could rescue colonic epithelial (HT-29) cells from apoptosis induced by proapoptotic cytokines that are increased during inflammatory bowel disease. The PAR2 agonists 2-furoyl-LIGRLO (2f-LI), SLIGKV and trypsin all significantly reduced cleavage of caspase-3, -8, and -9, poly(ADP-ribose) polymerase, and the externalization of phosphatidylserine after treatment of cells with IFN-γ and TNF-α. Knockdown of PAR2 with siRNA eliminated the anti-apoptotic effect of 2f-LI and increased the sensitivity of HT-29 cells to cytokine-induced apoptosis. Concurrent inhibition of both MEK1/2 and PI3K was necessary to inhibit PAR2-induced survival. 2f-LI was found to increase phosphorylation and inactivation of pro-apoptotic BAD at Ser(112) and Ser(136) by MEK1/2 and PI3K-dependent signaling, respectively. PAR2 activation also increased the expression of anti-apoptotic MCL-1. Simultaneous knockdown of both BAD and MCL-1 had minimal effects on PAR2-induced survival, whereas single knockdown had no effect. We conclude that PAR2 activation reduces cytokine-induced epithelial apoptosis via concurrent stimulation of MEK1/2 and PI3K but little involvement of MCL-1 and BAD. Our findings represent a novel mechanism whereby serine proteinases facilitate epithelial cell survival and may be important in the context of colonic healing.
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http://dx.doi.org/10.1074/jbc.M114.610485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4256365PMC
December 2014

Targeting mitochondria-derived reactive oxygen species to reduce epithelial barrier dysfunction and colitis.

Am J Pathol 2014 Sep 14;184(9):2516-27. Epub 2014 Jul 14.

Gastrointestinal Research Group, Department of Physiology and Pharmacology, Calvin, Phoebe, and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada; Inflammation Research Network, Department of Physiology and Pharmacology, Calvin, Phoebe, and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada. Electronic address:

Epithelial permeability is often increased in inflammatory bowel diseases. We hypothesized that perturbed mitochondrial function would cause barrier dysfunction and hence epithelial mitochondria could be targeted to treat intestinal inflammation. Mitochondrial dysfunction was induced in human colon-derived epithelial cell lines or colonic biopsy specimens using dinitrophenol, and barrier function was assessed by transepithelial flux of Escherichia coli with or without mitochondria-targeted antioxidant (MTA) cotreatment. The impact of mitochondria-targeted antioxidants on gut permeability and dextran sodium sulfate (DSS)-induced colitis in mice was tested. Mitochondrial superoxide evoked by dinitrophenol elicited significant internalization and translocation of E. coli across epithelia and control colonic biopsy specimens, which was more striking in Crohn's disease biopsy specimens; the mitochondria-targeted antioxidant, MitoTEMPO, inhibited these barrier defects. Increased gut permeability and reduced epithelial mitochondrial voltage-dependent anion channel expression were observed 3 days after DSS. These changes and the severity of DSS-colitis were reduced by MitoTEMPO treatment. In vitro DSS-stimulated IL-8 production by epithelia was reduced by MitoTEMPO. Metabolic stress evokes significant penetration of commensal bacteria across the epithelium, which is mediated by mitochondria-derived superoxide acting as a signaling, not a cytotoxic, molecule. MitoTEMPO inhibited this barrier dysfunction and suppressed colitis in DSS-colitis, likely via enhancing barrier function and inhibiting proinflammatory cytokine production. These novel findings support consideration of MTAs in the maintenance of epithelial barrier function and the management of inflammatory bowel diseases.
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http://dx.doi.org/10.1016/j.ajpath.2014.05.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4188172PMC
September 2014

MicroRNA-34a mediates the autocrine signaling of PAR2-activating proteinase and its role in colonic cancer cell proliferation.

PLoS One 2013 26;8(8):e72383. Epub 2013 Aug 26.

State Key Laboratory of Molecular Oncology, Cancer Institute/Cancer Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China.

The tumor microenvironment is replete with proteinases. As a sensor of proteinases, proteinase activated receptor 2 (PAR2) plays critical roles in tumorigenesis. We showed that PAR2 and its activating proteinase were coexpressed in different colon cancer cell lines, including HT29. Inactivating proteinase or knockdown of PAR2 significantly not only reduced cell proliferation in vitro but also inhibited tumorigenicity of HT29 in vivo. In addition, activation of PAR2 promoted DNA synthesis and upregulated Cyclin D1 activity at both transcriptional and post-transcriptional levels. Further studies showed that miRNA-34a mediated PAR2-induced Cyclin D1 upregulation. Inhibition of miR-34a partially abolished the suppression of Cyclin D1 induced by PAR2 deficiency. In addition, we showed that TGF-β contributed to the regulation of miR-34a by PAR2. Finally, in colorectal carcinoma samples, upregulation of PAR2 and downregulation of miR-34a were significantly correlated with grade and lymphomatic metastasis. Our findings provide the first evidence that miRNA mediates autocrine proteinase signaling-mediated cancer cell proliferation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072383PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753253PMC
June 2014

Intrarectal instillation of Clostridium difficile toxin A triggers colonic inflammation and tissue damage: development of a novel and efficient mouse model of Clostridium difficile toxin exposure.

Infect Immun 2012 Dec 8;80(12):4474-84. Epub 2012 Oct 8.

Department of Medicine, University of Calgary, Calgary, Canada.

Clostridium difficile, a major cause of hospital-acquired diarrhea, triggers disease through the release of two toxins, toxin A (TcdA) and toxin B (TcdB). These toxins disrupt the cytoskeleton of the intestinal epithelial cell, increasing intestinal permeability and triggering the release of inflammatory mediators resulting in intestinal injury and inflammation. The most prevalent animal model to study TcdA/TcdB-induced intestinal injury involves injecting toxin into the lumen of a surgically generated "ileal loop." This model is time-consuming and exhibits variability depending on the expertise of the surgeon. Furthermore, the target organ of C. difficile infection (CDI) in humans is the colon, not the ileum. In the current study, we describe a new model of CDI that involves intrarectal instillation of TcdA/TcdB into the mouse colon. The administration of TcdA/TcdB triggered colonic inflammation and neutrophil and macrophage infiltration as well as increased epithelial barrier permeability and intestinal epithelial cell death. The damage and inflammation triggered by TcdA/TcdB isolates from the VPI and 630 strains correlated with the concentration of TcdA and TcdB produced. TcdA/TcdB exposure increased the expression of a number of inflammatory mediators associated with human CDI, including interleukin-6 (IL-6), gamma interferon (IFN-γ), and IL-1β. Finally, we were able to demonstrate that TcdA was much more potent at inducing colonic injury than was TcdB but TcdB could act synergistically with TcdA to exacerbate injury. Taken together, our data indicate that the intrarectal murine model provides a robust and efficient system to examine the effects of TcdA/TcdB on the induction of inflammation and colonic tissue damage in the context of human CDI.
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http://dx.doi.org/10.1128/IAI.00933-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497439PMC
December 2012

Epidermal growth factor receptor transactivation is required for proteinase-activated receptor-2-induced COX-2 expression in intestinal epithelial cells.

Am J Physiol Gastrointest Liver Physiol 2012 Jul 19;303(1):G111-9. Epub 2012 Apr 19.

Dept. of Physiology and Pharmacology, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB, Canada.

Proteinase-activated receptor (PAR)(2), a G protein-coupled receptor activated by serine proteinases, has been implicated in both intestinal inflammation and epithelial proliferation. Cyclooxygenase (COX)-2 is overexpressed in the gut during inflammation as well as in colon cancer. We hypothesized that PAR(2) drives COX-2 expression in intestinal epithelial cells. Treatment of Caco-2 colon cancer cells with the PAR(2)-activating peptide 2-furoyl-LIGRLO-NH(2) (2fLI), but not by its reverse-sequence PAR(2)-inactive peptide, for 3 h led to an increase in intracellular COX-2 protein expression accompanied by a COX-2-dependent increase in prostaglandin E(2) production. 2fLI treatment for 30 min significantly increased metalloproteinase activity in the culture supernatant. Increased epidermal growth factor receptor (EGFR) phosphorylation was observed in cell lysates following 40 min of treatment with 2fLI. The broad-spectrum metalloproteinase inhibitor marimastat inhibited both COX-2 expression and EGFR phosphorylation. The EGFR tyrosine kinase inhibitor PD153035 also abolished 2fLI-induced COX-2 expression. Although PAR(2) activation increased ERK MAPK phosphorylation, neither ERK pathway inhibitors nor a p38 MAPK inhibitor affected 2fLI-induced COX-2 expression. However, inhibition of either Src tyrosine kinase signaling by PP2, Rho kinase signaling by Y27632, or phosphatidylinositol 3 (PI3) kinase signaling by LY294002 prevented 2fLI-induced COX-2 expression. Trypsin increased COX-2 expression through PAR(2) in Caco-2 cells and in an EGFR-dependent manner in the noncancerous intestinal epithelial cell-6 cell line. In conclusion, PAR(2) activation drives COX-2 expression in Caco-2 cells via metalloproteinase-dependent EGFR transactivation and activation of Src, Rho, and PI3 kinase signaling. Our findings provide a mechanism whereby PAR(2) can participate in the progression from chronic inflammation to cancer in the intestine.
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http://dx.doi.org/10.1152/ajpgi.00358.2011DOI Listing
July 2012

Escherichia coli-induced epithelial hyporesponsiveness to secretagogues is associated with altered CFTR localization.

Cell Microbiol 2012 Apr 8;14(4):447-59. Epub 2012 Feb 8.

Department of Physiology and Pharmacology, Inflammation Research Group, University of Calgary, Calgary, AB T2N 4N1, Canada.

Both pathogenic and commensal strains of Escherichia coli colonize the human intestinal tract. Pathogenic strains differ only in the expression of virulence factors, many of which comprise a type III secretion system (TTSS). Little is known regarding the effect of E. coli on the intestinal epithelial response to the secretagogues that drive ion secretion, despite its importance in causing clinically significant diarrhoea. Using Ussing chambers to measure electrogenic ion transport of T84 intestinal epithelial cell monolayers, we found that all strains of E. coli tested (pathogenic, commensal, probiotic and lab strain) significantly reduced cAMP-dependent ion secretion after 4-8 h exposure. Enteropathogenic E. coli mutants lacking a functional TTSS caused similar hyposecretion while not causing significant apoptosis (as shown by caspase-3 cleavage) or necrosis (lactate dehydrogenase release), as did the commensal strain F18, indicating that epithelial cell death was not the cause of hyposecretion. Enteropathogenic E. coli and the TTSS mutant significantly reduced cell surface expression of the apical anion channel, cystic fibrosis transmembrane conductance regulator, which is likely the mechanism behind the pathogen-induced hyposecretion. However, F18 did not cause cystic fibrosis transmembrane conductance regulator mislocalization and the commensal-induced mechanism remains unclear.
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http://dx.doi.org/10.1111/j.1462-5822.2011.01744.xDOI Listing
April 2012

Salvinorin A has antiinflammatory and antinociceptive effects in experimental models of colitis in mice mediated by KOR and CB1 receptors.

Inflamm Bowel Dis 2012 Jun 26;18(6):1137-45. Epub 2011 Sep 26.

Snyder Institute of Infection, Immunity and Inflammation (III), University of Calgary, AB, Canada.

Background: Salvinorin A (SA) has a potent inhibitory action on mouse gastrointestinal (GI) motility and ion transport, mediated primarily by kappa-opioid receptors (KOR). The aim of the present study was to characterize possible antiinflammatory and antinociceptive effects of SA in the GI tract of mice.

Methods: Colonic damage scores and myeloperoxidase activity were determined after intraperitoneal (i.p.), intracolonic (i.c.), and oral (p.o.) administration of SA using the trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) models of colitis in mice. Additionally, KOR, cannabinoid (CB)1, and CB2 western blot analysis of colon samples was performed. The antinociceptive effect of SA was examined based on the number of behavioral responses to i.c. instillation of mustard oil (MO).

Results: The i.p. (3 mg/kg, twice daily) and p.o. (10 mg/kg, twice daily) administration of SA significantly attenuated TNBS and DSS colitis in mice. The effect of SA was blocked by KOR antagonist nor-binaltorphimine (10 mg/kg, i.p.). Western blot analysis showed no influence of SA on KOR, CB1, or CB2 levels. SA (3 mg/kg, i.p. and 10 mg/kg, i.c.) significantly decreased the number of pain responses after i.c. instillation of MO in the vehicle- and TNBS-treated mice. The antinociceptive action of SA was blocked by KOR and CB1 antagonists. The analgesic effect of i.c. SA was more potent in TNBS-treated mice compared to controls.

Conclusions: Our results suggest that the drugs based on the structure of SA have the potential to become valuable antiinflammatory or analgesic therapeutics for the treatment of GI diseases.
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http://dx.doi.org/10.1002/ibd.21873DOI Listing
June 2012

Probiotic bacteria and intestinal epithelial barrier function.

Am J Physiol Gastrointest Liver Physiol 2010 Jun 18;298(6):G807-19. Epub 2010 Mar 18.

Department of Physiology and Pharmacology, Univ. of Calgary, 3330 Hospital Dr. NW, Calgary, AB, Canada T2N 4N1.

The intestinal tract is a diverse microenvironment where more than 500 species of bacteria thrive. A single layer of epithelium is all that separates these commensal microorganisms and pathogens from the underlying immune cells, and thus epithelial barrier function is a key component in the arsenal of defense mechanisms required to prevent infection and inflammation. The epithelial barrier consists of a dense mucous layer containing secretory IgA and antimicrobial peptides as well as dynamic junctional complexes that regulate permeability between cells. Probiotics are live microorganisms that confer benefit to the host and that have been suggested to ameliorate or prevent diseases including antibiotic-associated diarrhea, irritable bowel syndrome, and inflammatory bowel disease. Probiotics likely function through enhancement of barrier function, immunomodulation, and competitive adherence to the mucus and epithelium. This review summarizes the evidence about effects of the many available probiotics with an emphasis on intestinal barrier function and the mechanisms affected by probiotics.
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http://dx.doi.org/10.1152/ajpgi.00243.2009DOI Listing
June 2010

Proteinase-activated receptors induce interleukin-8 expression by intestinal epithelial cells through ERK/RSK90 activation and histone acetylation.

FASEB J 2010 Jun 11;24(6):1971-80. Epub 2010 Jan 11.

Inflammation Research Network, University of Calgary, Calgary, AB, Canada.

Proteinase-activated receptors (PARs) are involved in both inflammation and tumorigenesis in epithelial cells. Interleukin (IL)-8 is a potent chemoattractant and is also involved in angiogenesis. The molecular mechanism whereby PARs induce epithelial IL-8 expression is not known. In HT-29 colonic epithelial cells, PAR(1) or PAR(2) agonists stimulated the expression of IL-8 through a NF-kappaB-dependent pathway without inducing IkappaB degradation and disassociation of IkappaB from NF-kappaB. Further studies revealed that PAR activation induced the phosphorylation of p65 at Ser-276 in the nucleus, which increased the recruitment of histone acetyltransferase (HAT) p300 to p50. Inhibition of ERK activation completely blocked PAR-induced IL-8 expression, phosphorylation of p65 and HAT activity. We also demonstrated that RSK p90 was the downstream kinase that mediated ERK-induced nuclear p65 phosphorylation. In conclusion, activation of either PAR(1) or PAR(2) stimulated the transcriptional up-regulation of IL-8 in HT-29 colonic epithelial cells through a pathway that involved ERK/RSK p90, NF-kappaB phosphorylation, and HAT activity. These studies provide evidence of a new role for serine proteinases and PARs in the regulation of gene expression in colonic inflammation and tumorigenesis.
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http://dx.doi.org/10.1096/fj.09-137646DOI Listing
June 2010

Serine proteases decrease intestinal epithelial ion permeability by activation of protein kinase Czeta.

Am J Physiol Gastrointest Liver Physiol 2009 Jul 21;297(1):G60-70. Epub 2009 May 21.

Inflammation Research Network, Department of Physiology and Pharmacology, University of Calgary, Calgary T2N 4N1, Canada.

Epithelial permeability to ions and larger molecules in the gut is essential for fluid balance, and its dysregulation contributes to intestinal pathology. We investigated the effect of digestive serine proteases on epithelial paracellular permeability. Trypsin, chymotrypsin, and elastase elicited sustained increases in transepithelial resistance (R(TE)) in polarized monolayers of three intestinal epithelial cell lines. This effect was reflected by decreases in paracellular conductances of Na+ and Cl- and a concomitant decrease in permeability to 3,000 molecular weight dextran. The enzyme activities of the proteases were required, yet activators of known protease-activated receptors (PARs) did not reproduce the effect of these proteases on R(TE). PKCzeta isoform-specific inhibitor significantly reduced the trypsin-induced increase in R(TE) whereas PKCzeta activity was increased in cells treated with trypsin and chymotrypsin compared with control cells; this activity was reduced to control levels in the presence of PKCzeta-specific inhibitor. Ca2+ chelators and pharmacological inhibitors of cell signaling support the role for PKCzeta in the protease-induced effect. Finally, we showed that treatment with the serine proteases increased occludin immunostaining and zonula occludin-1 coimmunoprecipitation with occludin in the detergent-insoluble fraction of cell lysates, and these increases were ablated by pretreatment with PKCzeta-specific inhibitor. This finding indicates increased insertion of occludin into the cell junctional complex. These data demonstrate a role for serine proteases in the facilitation of epithelial barrier function through a mechanism that is independent of PARs and is mediated by activation of PKCzeta.
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http://dx.doi.org/10.1152/ajpgi.00096.2009DOI Listing
July 2009