Christel Salvador-Cartier

PAR2 activation alters colonic paracellular permeability in mice via IFN-γ-dependent and -independent pathways

Activation of colonic proteinase‐activated receptor‐2 (PAR2) caused inflammation and increased mucosal permeability in mouse colon. The present study was aimed at characterizing the possible links between these two phenomena. We evaluated the effects of intracolonic infusion of PAR2‐activating peptide, SLIGRL, on colonic paracellular permeability and inflammation at two different doses, 5 and 100 μg per mouse, in an attempt to discriminate between both PAR2‐mediated effects. We further investigated the possible involvement of interferon γ (IFN‐γ) and calmodulin‐dependent activation of myosin light chain kinase (MLCK), and alterations of zonula occludens‐1 (ZO‐1) localization in PAR2‐induced responses. Thus, at the lower dose, SLIGRL increased colonic permeability without causing inflammation. Western blotting showed phosphorylation of mucosal myosin light chain (MLC) expression after both doses of SLIGRL. Moreover, while the MLCK inhibitor, ML‐7, abolished the permeability effects of the low dose of SLIGRL, it only partially inhibited that of the high dose. In IFN‐γ‐deficient mice (B6 ifng−/−), the increases in permeability were similar for both doses of SLIGRL and prevented by ML‐7. In addition, MLCK immunoprecipitation revealed an increase of calmodulin binding to MLCK in the mucosa of mice treated with either dose of SLIGRL. Finally, we have shown that direct activation of PAR2 on enterocytes is responsible for increased permeability and ZO‐1 disruption. Moreover, SLIGRL at a dose that does not produce inflammation increases permeability via calmodulin activation, which binds and activates MLCK. The resulting tight junction opening does not depend upon IFN‐γ secretion, while the increased permeability in response to the high dose of PAR2 agonist involves IFN‐γ secretion.

Myosin light chain kinase is involved in lipopolysaccharide-induced disruption of colonic epithelial barrier and bacterial translocation in rats.

Sepsis is associated with bacterial translocation (BT) and changes in colonic paracellular permeability (CPP), but the link between these effects is unknown. The present study aimed to identify whether changes in CPP after lipopolysaccharide (LPS) administration triggers BT, colonic inflammation, visceral pain, and sickness behavior and to evaluate the role of myosin light chain kinase (MLCK) in colonocyte cytoskeleton contraction. Rats received the MLCK inhibitor ML-7 alone or combined with LPS. CPP was measured for 6 hours after administration. Visceral pain, food intake, BT, electron microscopy of tight junctions of colonocytes, cytokine levels, and Western blotting of phosphorylated MLC from colonic mucosa were assessed in a time range of 0 to 3 hours after treatment. Sepsis increased CPP at 0 to 6 hours after LPS and associated with tight junction morphological changes, increased MLC phosphorylation, and mucosal release of proinflammatory cytokines. Massive BT, visceral hyperalgesia, and reduced food intake were also observed. Addition of ML-7 prevented all LPS-induced effects, except for changes in food intake. In conclusion, LPS-mediated effects on CPP include gut inflammation, BT, and visceral hyperalgesia. Inhibition of MLCK-dependent colonocyte cytoskeleton contraction by ML-7 prevents the LPS-induced alterations of CPP and its subsequent effects.

Long term alterations of colonic nerve mast cell interactions induced by neonatal maternal deprivation in rats

Background: Neonatal maternal deprivation induces colonic alterations in adult rats, such as hypersensitivity to distension or an increase in paracellular permeability, characteristics of irritable bowel syndrome (IBS) patients. Recent studies described neuroimmune alterations in the colonic mucosa of IBS patients. Methods: Male Wistar rats were submitted to maternal deprivation for 3 h daily during postnatal days 2–14, and were sacrificed at 4 or 12 weeks of age. Control pups were left undisturbed with their dam. Results: Colonic mast cell hyperplasia was observed at 4 and 12 weeks in maternally deprived rats, and was associated with an increase in protease content. Mucosal nerve fibre density assessed by protein gene product (PGP) 9.5 immunoreactivity was increased at 12 weeks but not at 4 weeks, while calcitonin gene-related protein (CGRP)-immunoreactive fibres remain constant. Synaptogenesis assessed by synaptophysin immunostaining was increased at 4 weeks but not at 12 weeks. The number of mast cells in close proximity to PGP 9.5- or CGRP-immunoreactive fibres was greater at both 4 and 12 weeks. Expression of neurokinin NK1 receptors in the spinal cord was enhanced at 12 weeks. No significant change in total mast cell number, PGP 9.5 immunoreactivity and mast cells associated with PGP 9.5-immunoreactive fibres was observed in the jejunum. Treatment of pups with anti-nerve growth factor (NGF) antibodies abolished the increases in synaptogenesis and in the number of mast cells in close proximity to nerve fibres observed 4 weeks after maternal deprivation. Conclusions: Neonatal maternal deprivation induces closer association of colonic mast cells with nerves, which is similar to that seen in IBS patients. NGF is a possible mediator of this effect.

Phenotypic changes in colonocytes following acute stress or activation of mast cells in mice: implications for delayed epithelial barrier dysfunction

Background and aim: Stressful life events are known to modulate the development or relapse of disease in both inflammatory bowel disease and irritable bowel disease patients but underlying mechanisms remain unclear. Stress is known to effect mast cells, interferon γ (IFN-γ), and myosin light chain phosphorylation to trigger colonic epithelial barrier dysfunction. The aim of this study was to investigate whether acute stress induced or chemical mast cell activation impaired expression and function of epithelial tight junctions, and altered colonocyte differentiation in mice. Methods: Colonic paracellular permeability was assessed as the in vivo lumen to blood ratio of 51Cr-EDTA in different groups of mice (controls, stressed, mast cell degranulator BrX-537A treated), pretreated or not with the mast cell stabiliser doxantrazole. Involvement of mast cells and IFN-γ was evaluated in wild-type and IFN-γ deficient mice. Tight junction alteration was assessed by histology, transmission electron microscopy, and real time reverse transcription-polymerase chain reaction. Colonocyte differentiation was determined by protein kinase C ζ (PKCζ) immunofluorescence and western blotting, and alkaline phosphatase activity assay. Results: Acute stress induced a three day delayed increase in colonic paracellular permeability which involved mast cell degranulation and overproduction of IFN-γ. The colonic epithelial barrier was morphologically altered and expression of mRNA encoding tight junction proteins ZO-2 and occludin was decreased. Moreover, three days after acute stress, colonocyte differentiation was reduced, as shown by decreased expression of both PKCζ isotype and alkaline phosphatase. Conclusion: These data highlight new mechanisms whereby an acute stress acts on the gastrointestinal tract by inducing alterations in colonocyte differentiation and decreased expression of mRNA encoding tight junction proteins. Thus phenotypic changes in colonocytes could pave the way for stress related intestinal disorders.

Improvement of an experimental colitis in rats by lactic acid bacteria producing superoxide dismutase

&NA; The use of superoxide dismutases (SODs) in inflammatory diseases is hampered by their short circulatory half‐life. To determine whether a bacterial supply of SOD into the colon might improve an experimental colitis, the effects of oral treatment with live recombinant lactic acid bacteria producing different amounts of SOD and those of colonic infusion of SOD were compared. Wistar rats were fitted with a catheter in the proximal colon through which TNBS was administered to induce colitis. Animals received a continuous intracolonic infusion of bovine SOD (40 U per rat per day) for 4 days after TNBS or were treated orally with live recombinant Lactococcus lactis or Lactobacillus plantarum strains (109 colony‐forming units (CFU)/d), producing or not producing SOD, for 4 days before and after TNBS. SOD activity of bacterial extracts was 0, 26, 74, and 624 units/109 CFU for L. plantarum, L. lactis, L. lactis SOD+, and L. plantarum SOD+, respectively. Four days after TNBS, macroscopic and microscopic damage, myeloperoxidase (MPO) activity, and nitrotyrosine immunostaining were evaluated. TNBS induced macroscopic and microscopic damages, an increase in MPO activity, and intense immunostaining for nitrotyrosine. Macroscopic damage and MPO activity were reduced by bovine SOD. These parameters and microscopic damages also were reduced by L. lactis, L. lactis SOD+, and L. plantarum SOD+, but not by L. plantarum. Nitrotyrosine immunostaining was attenuated after treatment with the 4 bacterial strains. Although not all of the anti‐inflammatory effects could be attributed directly to SOD, our results suggest that SOD‐producing lactic acid bacteria open a novel approach in inflammatory bowel disease treatment.

Genotoxicity of Escherichia coli Nissle 1917 strain cannot be dissociated from its probiotic activity.

Oral administration of the probiotic bacterium Escherichia coli Nissle 1917 improves chronic inflammatory bowel diseases, but the molecular basis for this therapeutic efficacy is unknown. E. coli Nissle 1917 harbors a cluster of genes coding for the biosynthesis of hybrid nonribosomal peptide-polyketide(s). This biosynthetic pathway confers the ability for bacteria to induce DNA double strand breaks in eukaryotic cells. Here we reveal that inactivation of the clbA gene within this genomic island abrogated the ability for the strain to induce DNA damage and chromosomal abnormalities in non-transformed cultured rat intestinal epithelial cells but is required for the probiotic activity of E. coli Nissle 1917. Thus, evaluation of colitis severity induced in rodent fed with E. coli Nissle 1917 or an isogenic non-genotoxic mutant demonstrated the need for a functional biosynthetic pathway both in the amelioration of the disease and in the modulation of cytokine expression. Feeding rodents with a complemented strain for which genotoxicity was restored confirmed that this biosynthetic pathway contributes to the health benefits of the probiotic by modulating its immunomodulatory properties. Our data provide additional evidence for the benefit of this currently used probiotic in colitis but remind us that an efficient probiotic may also have side effects as any other medication.

Colonic luminal proteases activate colonocyte proteinase-activated receptor-2 and regulate paracellular permeability in mice.

Abstract  Luminal activation of protease‐activated receptors‐2 (PAR2) on colonocytes by trypsin or PAR2‐activating peptide increases colonic paracellular permeability (CPP). The aim of this study was to evaluate the role of proteases from endogenous and bacterial origin in the modulation of CPP and colonocyte PAR2 expression in mice. CPP was assessed with 51Cr‐EDTA after intracolonic administration of different protease inhibitors. After 12 days of oral antibiotic treatment, measurements of colonic luminal serine protease activity (CLSPA), CPP, mucosal mouse mast cell proteinase‐1 (MMCP‐1) content, immunochemistry of PAR2 and assessment of effects of PAR2 agonist (SLIGRL) and mast cell degranulator (C48/80) on CPP in Ussing chambers were performed. Immunochemistry was repeated after intracolonic trypsin administration. Colonic infusion of protease inhibitors significantly reduced CPP. In antibiotic‐treated mice, CLSPA was reduced coupled with a decrease in PAR2 expression, but with no change in CPP and MMCP‐1 content. Trypsin administration restored PAR2 expression. The increase in CPP induced by SLIGRL and C48/80 was reduced after antibiotic treatment. Protease activity of colonic content plays an important role in the regulation of mucosal barrier through activation of PAR2.

Stress disrupts intestinal mucus barrier in rats via mucin O-glycosylation shift: prevention by a probiotic treatment.

Despite well-known intestinal epithelial barrier impairment and visceral hypersensitivity in irritable bowel syndrome (IBS) patients and IBS-like models, structural and physical changes in the mucus layer remain poorly understood. Using a water avoidance stress (WAS) model, we aimed at evaluating whether 1) WAS modified gut permeability, visceral sensitivity, mucin expression, biochemical structure of O-glycans, and related mucus physical properties, and 2) whether Lactobacillus farciminis treatment prevented these alterations. Wistar rats received orally L. farciminis or vehicle for 14 days; at day 10, they were submitted to either sham or 4-day WAS. Intestinal paracellular permeability and visceral sensitivity were measured in vivo. The number of goblet cells and Muc2 expression were evaluated by histology and immunohistochemistry, respectively. Mucosal adhesion of L. farciminis was determined ex situ. The mucin O-glycosylation profile was obtained by mass spectrometry. Surface imaging of intestinal mucus was performed at nanoscale by atomic force microscopy. WAS induced gut hyperpermeability and visceral hypersensitivity but did not modify either the number of intestinal goblet cells or Muc2 expression. In contrast, O-glycosylation of mucins was strongly affected, with the appearance of elongated polylactosaminic chain containing O-glycan structures, associated with flattening and loss of the mucus layer cohesive properties. L. farciminis bound to intestinal Muc2 and prevented WAS-induced functional alterations and changes in mucin O-glycosylation and mucus physical properties. WAS-induced functional changes were associated with mucus alterations resulting from a shift in O-glycosylation rather than from changes in mucin expression. L. farciminis treatment prevented these alterations, conferring epithelial and mucus barrier strengthening.

Dexamethasone prevents visceral hyperalgesia but not colonic permeability increase induced by luminal protease - activated receptor-2 agonist in rats

Background: Low-grade inflammation may play a role in the pathogenesis of irritable bowel syndrome (IBS). Although corticosteroids are potent inhibitors of inflammatory processes, only one study with corticosteroids in patients with postinfectious IBS exists, which suggests that prednisolone is not an effective treatment for IBS symptoms. Aim: To evaluate whether dexamethasone treatment prevents protease-activated receptor-2 (PAR-2) activation-induced visceral hyperalgesia and increased permeability in rats, and to determine whether the effects involve colonic mast cells. Methods: Abdominal contractions provoked by rectal distension were recorded in rats equipped with intramuscular electrodes. Changes in visceral hypersensitivity provoked by intracolonic administration of PAR-2-activating peptide (SLIGRL; H-serine-leucine-isoleucine-glycine-arginine-leucine-OH), changes in colonic mucosal rat mast cell protease-II (RMCP-II) content, mast cell count and PAR-2 expression were measured after a 4-day treatment with dexamethasone (1 mg/day/rat intraperitoneally) or its vehicle (water). The effect of mast cell stabiliser (doxantrazole, 1 mg/kg intraperitoneally, 2 h before and 6 h after intracolonic infusion of SLIGRL) on SLIGRL-induced visceral hyperalgesia was also assessed. The effects of SLIGRL and a mast cell degranulator (compound 48/80) on the permeability of colonic strips from vehicle- or dexamethasone-treated rats were investigated in Ussing chambers. Results: 4 days of dexamethasone as well as doxantrazole diminished the SLIGRL-induced hyperalgesia for all volumes of distension. This effect of dexamethasone was accompanied by a reduced responsiveness of colonic permeability to compound 48/80, and decreased RMCP-II content and mast cell number. Dexamethasone treatment did not influence colonic mucosal PAR-2 expression and permeability responsiveness to SLIGRL. Conclusions: Dexamethasone treatment improves PAR-2 agonist-induced visceral hypersensitivity but does not prevent PAR-2 agonist-induced increase in colonic permeability in rats. This effect is coupled with a reduction of colonic mast cell number and RMCP-II contents.

A Low Dose of Fermented Soy Germ Alleviates Gut Barrier Injury, Hyperalgesia and Faecal Protease Activity in a Rat Model of Inflammatory Bowel Disease

Pro-inflammatory cytokines like macrophage migration inhibitory factor (MIF), IL-1β and TNF-α predominate in inflammatory bowel diseases (IBD) and TNBS colitis. Increased levels of serine proteases activating protease-activated receptor 2 (PAR-2) are found in the lumen and colonic tissue of IBD patients. PAR-2 activity and pro-inflammatory cytokines impair epithelial barrier, facilitating the uptake of luminal aggressors that perpetuate inflammation and visceral pain. Soy extracts contain phytoestrogens (isoflavones) and serine protease inhibitors namely Bowman-Birk Inhibitors (BBI). Since estrogens exhibit anti-inflammatory and epithelial barrier enhancing properties, and that a BBI concentrate improves ulcerative colitis, we aimed to evaluate if a fermented soy germ extract (FSG) with standardized isoflavone profile and stable BBI content exert cumulative or synergistic protection based on protease inhibition and estrogen receptor (ER)-ligand activity in colitic rats. Female rats received orally for 15 d either vehicle or FSG with or without an ER antagonist ICI 182.780 before TNBS intracolonic instillation. Macroscopic and microscopic damages, myeloperoxidase activity, cytokine levels, intestinal paracellular permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression were assessed 24 h, 3 d and 5 d post-TNBS. FSG treatment improved the severity of colitis, by decreasing the TNBS-induced rise in gut permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression at all post-TNBS points. All FSG effects were reversed by the ICI 182.780 except the decrease in faecal proteolytic activity and PAR-2 expression. In conclusion, the anti-inflammatory properties of FSG treatment result from two distinct but synergic pathways i.e an ER-ligand and a PAR-2 mediated pathway, providing rationale for potential use as adjuvant therapy in IBD.