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Background & Aims: Separation of newborn rats from their mothers induces visceral hypersensitivity and impaired epithelial secretory cell lineages when they are adults. Little is known about the mechanisms by which maternal separation causes visceral hypersensitivity or its relationship with defects in epithelial secretory cell lineages., Methods: We performed studies with C3H/HeN mice separated from their mothers as newborns and mice genetically engineered (Sox9 flox/flox -vil-cre on C57BL/6 background) to have deficiencies in Paneth cells. Paneth cell deficiency was assessed by lysozyme staining of ileum tissues and lysozyme activity in fecal samples. When mice were 50 days old, their abdominal response to colorectal distension was assessed by electromyography. Fecal samples were collected and microbiota were analyzed using Gut Low-Density Array quantitative polymerase chain reaction., Results: Mice with maternal separation developed visceral hypersensitivity and defects in Paneth cells, as reported from rats, compared with mice without maternal separation. Sox9 flox/flox -vil-Cre mice also had increased visceral hypersensitivity compared with control littermate Sox9 flox/flox mice. Fecal samples from mice with maternal separation and from Sox9 flox/flox -vil-cre mice had evidence for intestinal dysbiosis of the microbiota, characterized by expansion of Escherichia coli. Daily gavage of conventional C3H/HeN adult mice with 10 9 commensal E coli induced visceral hypersensitivity. Conversely, daily oral administration of lysozyme prevented expansion of E coli during maternal separation and visceral hypersensitivity., Conclusions: Mice with defects in Paneth cells (induced by maternal separation or genetically engineered) have intestinal expansion of E coli leading to visceral hypersensitivity. These findings provide evidence that Paneth cell function and intestinal dysbiosis are involved in visceral sensitivity., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The present study aimed at detecting the exogenously applied probiotic Lactobacillus farciminis in rats, after exposure to IBS-like chronic stress, based on 4-day Water Avoidance Stress (WAS). The presence of L. farciminis in both ileal and colonic mucosal tissues was demonstrated by FISH and qPCR, with ileum as the preferential niche, as for the SFB population. A different spatial distribution of the probiotic was observed: in the ileum, bacteria were organized in micro-colonies more or less close to the epithelium whereas, in the colon, they were mainly visualized far away from the epithelium. When rats were submitted to WAS, the L. farciminis population substantially decreased in both intestinal regions, due to a stress-induced increase in colonic motility and defecation, rather than a modification of bacterial binding to the intestinal mucin Muc2.

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., (Copyright © 2014 the American Physiological Society.)

The neonatal gut is rapidly colonized by a newly dominant group of commensal Escherichia coli strains among which a large proportion produces a genotoxin called colibactin. In order to analyze the short- and long-term effects resulting from such evolution, we developed a rat model mimicking the natural transmission of E. coli from mothers to neonates. Genotoxic and non-genotoxic E. coli strains were equally transmitted to the offspring and stably colonized the gut across generations. DNA damage was only detected in neonates colonized with genotoxic E. coli strains. Signs of genotoxic stress such as anaphase bridges, higher occurrence of crypt fission and accelerated renewal of the mature epithelium were detected at adulthood. In addition, we observed alterations of secretory cell populations and gut epithelial barrier. Our findings illustrate how critical is the genotype of E. coli strains acquired at birth for gut homeostasis at adulthood.

Background & Aims: Irritable bowel syndrome (IBS) often associated with psychological distress, is characterized by increased gut permeability and visceral sensitivity. In animals, stress increases intestinal paracellular permeability (IPP), visceral sensitivity and colonic proteolytic activity. Estradiol reduces IPP and affects visceral sensitivity in non-stressed ovariectomized rats, but whether estrogens affect stress-induced hyperpermeability and hypersensitivity in cyclic females remains unclear. We aimed to evaluate (i) the effects of a phytoestrogen-rich soy germ fermented ingredient (SG) on visceral hypersensitivity, hyperpermeability and other symptoms in stressed intact female rats, (ii) the mechanisms of action involved on the basis of both estrogenic and protease inhibitor activities of SG., Methods: Female rats received orally for 15-d either SG, 17β-estradiol benzoate (EB), or vehicles, with or without the estrogen receptor (ER) antagonist ICI182.780 before stress. Visceral sensitivity, IPP, faecal proteolytic activity, plasma corticosterone, rat mast cell protease II immunostaining, and occludin expression were assessed., Results: Stress increased IPP (concomitantly to a drop in occludin expression), visceral sensitivity, faecal proteolytic activity and plasma corticosterone. Similarly to EB, SG prevented the stress-induced hyperpermeability, and hypersensitivity, without changes in plasma corticosterone. SG inhibited the increase in faecal proteolytic activity, enhanced occludin expression, and reduced the colonic mast cell density. All SG effects, except decrease on faecal proteolytic activity, were blocked by ICI182.780., Conclusion: A 2-wk oral treatment with SG prevented the stress-induced hyperpermeability and visceral hypersensitivity in cyclic rats through ER activation, and blocked the increase in colonic proteolytic activity, suggesting that SG can be promising in IBS management., (Copyright © 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

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.

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.

LPS-induced endotoxemia is associated with gut immune stimulation, mucosal inflammation, colonic paracellular permeability (CPP) alteration, and it promotes bacterial translocation (BT). Gut permeability increase linked to LPS promotes mucosal barrier dysfunction resulting to BT. However, the mechanisms involved in these alterations remain unknown. We aimed to evaluate the role of colonic mucosal mast cells and luminal serine protease activity (PA) in the alterations of CPP and BT induced by LPS. Rats receiving doxantrazole, a mast cell stabilizer, combined or not with LPS from Escherichia coli and CPP as well as BT were evaluated after each treatment. Mucosal mast cell activation was assessed by histological methods and by rat mast cell protease 2 level measurement in colonic content. Colonic luminal PA and mucosal inflammation (myeloperoxidase activity) were biochemically determined. In addition, the ability of luminal contents to act on CPP was evaluated in vitro in Ussing chambers. Peripheral administration of LPS promoted mast cell degranulation and increased CPP, BT, mucosal myeloperoxidase activity as well as rat mast cell protease 2 levels, and PA in colonic content. LPS-induced CPP increase and BT were prevented by doxantrazole. In vitro, exposure of the apical side of colonic tissues with supernatants from colonic contents of LPS-treated rats increased CPP. This effect was blocked by the serine protease inhibitor soybean trypsin inhibitor. Our data bring evidence of a key role of mucosal mast cells in LPS-induced increase of CPP and BT through the release of serine proteases into the colonic lumen.

Background and Aims: Sex steroids influence IBD symptoms. Macrophage migration inhibitory factor (MIF), a target of sex steroids in other inflammatory models, promotes interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha release in colitis. We investigated whether estradiol and progesterone influence MIF, IL-1beta, and TNF-alpha production in experimental colitis., Methods: Colonic MIF, IL-1beta, and TNF-alpha levels were measured in cyclic and ovariectomized rats, with or without estradiol benzoate (EB) or progesterone (P) replacement. MIF distribution was assessed by immunohistochemistry. Cytokines, myeloperoxidase activity, macroscopic damage, and plasma corticosterone were assessed 24 hours after intrarectal trinitrobenzene sulfonic acid (TNBS), with and without neutralizing anti-MIF antibody. Effects of EB and P on myeloperoxidase activity and MIF concentration were also assessed at 7 days in dextran sulfate sodium-induced colitis., Results: Basal IL-1beta and TNF-alpha contents did not fluctuate during the estrous cycle, while MIF concentrations increased from estrus (estrogen dominance) to metestrus (P dominance; P < .05). EB and P treatment mimicked these effects in ovariectomized rats, and similarly altered MIF immunostaining. Progesterone dominance aggravated TNBS colitis in comparison with estrogen. Progesterone enhanced TNBS-induced MIF (P < .001) and TNF-alpha (P < .01) production, while EB decreased MIF (P < .01) and IL-beta levels (P < .01). Anti-MIF antibody prevented P-mediated up-regulation of TNF-alpha, improved TNBS colitis, and enhanced plasma corticosterone. At 7 days after dextran sulfate sodium, EB decreased myeloperoxidase activity and MIF concentration, while P had no effect., Conclusions: Estrogen decreases while progesterone increases MIF production in the female rat colon. Changes in basal MIF contents may affect colon susceptibility to inflammation, by modulating TNF-alpha and IL-1beta production during early stages of colitis.

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 (10 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/10 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.

The structural integrity of the Golgi complex is essential to its functions in the maturation, sorting, and transport of plasma membrane proteins. Previously, we demonstrated that in pancreatic duct CFPAC-1 cells, which express DeltaF508 CFTR (cystic fibrosis transmembrane conductance regulator), the intracellular trafficking of carbonic anhydrase IV (CA IV), a membrane protein involved in HCO(3)(-) secretion, was impaired. To determine whether these abnormalities were related to changes in the Golgi complex, we examined the ultrastructure and distribution of Golgi compartments with regard to the microtubule cytoskeleton in CFPAC-1 cells transfected or not with the wild-type CFTR. Ultrastructural and immunocytochemical analysis showed that in polarized CFPAC-1 cells, Golgi stacks were disconnected from one another and scattered throughout the cytoplasm. The colocalization of CA IV with markers of Golgi compartments indicated the ability of stacks to transfer this enzyme. This Golgi dispersal was associated with abnormal microtubule distribution and multiplicity of the microtubule-organizing centers (MTOCs). In reverted cells, the normalization of Golgi structure, microtubule distribution, and MTOC number was observed. These observations suggest that the entire biosynthetic/secretory pathway is disrupted in CFPAC-1 cells, which might explain the abnormal intracellular transport of CA IV. Taken together, these results point to the fact that the expression of DeltaF508 CFTR affects the integrity of the secretory pathway.

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.

The respiratory system is directly exposed to low levels of lipopolysaccharide (LPS), present as a contaminant on airborne particles. In cystic fibrosis, the prevailing data identify structural changes of the airway epithelium, as well as tight junction dilatation. This study was aimed at determining the contribution of myosin light chain kinase to maintaining airway epithelium barrier integrity in the lung inflammatory response to LPS in rats. The effects of the selective myosin light chain kinase inhibitor, 5-iodonaphthalene-1-sulphonyl-homopiperazine (ML-7), were evaluated: 1) on pulmonary inflammation and airway epithelium barrier permeability alterations induced by intra-tracheal LPS from Pseudomonas aeruginosa; and 2) on levels of the phosphorylated form of the myosin light chain, which is increased in a human airway epithelial cell line (NCI-H292) and tracheal tissue after LPS exposure. The results show that LPS increased airway epithelium barrier paracellular permeability and lung inflammation, and that pre-treatment with ML-7 inhibited both effects. This effect of ML-7 was associated with the inhibition of phosphorylated myosin light chain in both NCI-H292 cells and tracheal tissue. The data, obtained using in vivo and in vitro approaches, demonstrate a key role for myosin light chain kinase in lung inflammation, and suggest that myosin light chain kinase could be a potential target for novel drugs intended for relief of lung injury.

Activation of colonic proteinase-activated receptor-2 (PAR(2)) 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 PAR(2)-activating peptide, SLIGRL, on colonic paracellular permeability and inflammation at two different doses, 5 and 100 microg per mouse, in an attempt to discriminate between both PAR(2)-mediated effects. We further investigated the possible involvement of interferon gamma (IFN-gamma) and calmodulin-dependent activation of myosin light chain kinase (MLCK), and alterations of zonula occludens-1 (ZO-1) localization in PAR(2)-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-gamma-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 PAR(2) 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-gamma secretion, while the increased permeability in response to the high dose of PAR(2) agonist involves IFN-gamma secretion.