Nicole C. Roy

Regulation of Tight Junction Permeability by Intestinal Bacteria and Dietary Components

The human intestinal epithelium is formed by a single layer of epithelial cells that separates the intestinal lumen from the underlying lamina propria. The space between these cells is sealed by tight junctions (TJ), which regulate the permeability of the intestinal barrier. TJ are complex protein structures comprised of transmembrane proteins, which interact with the actin cytoskeleton via plaque proteins. Signaling pathways involved in the assembly, disassembly, and maintenance of TJ are controlled by a number of signaling molecules, such as protein kinase C, mitogen-activated protein kinases, myosin light chain kinase, and Rho GTPases. The intestinal barrier is a complex environment exposed to many dietary components and many commensal bacteria. Studies have shown that the intestinal bacteria target various intracellular pathways, change the expression and distribution of TJ proteins, and thereby regulate intestinal barrier function. The presence of some commensal and probiotic strains leads to an increase in TJ proteins at the cell boundaries and in some cases prevents or reverses the adverse effects of pathogens. Various dietary components are also known to regulate epithelial permeability by modifying expression and localization of TJ proteins.

Lactobacillus plantarum MB452 enhances the function of the intestinal barrier by increasing the expression levels of genes involved in tight junction formation

BackgroundIntestinal barrier function is important for preserving health, as a compromised barrier allows antigen entry and can induce inflammatory diseases. Probiotic bacteria can play a role in enhancing intestinal barrier function; however, the mechanisms are not fully understood. Existing studies have focused on the ability of probiotics to prevent alterations to tight junctions in disease models, and have been restricted to a few tight junction bridging proteins. No studies have previously investigated the effect of probiotic bacteria on healthy intestinal epithelial cell genes involved in the whole tight junction signalling pathway, including those encoding for bridging, plaque and dual location tight junction proteins. Alteration of tight junction signalling in healthy humans is a potential mechanism that could lead to the strengthening of the intestinal barrier, resulting in limiting the ability of antigens to enter the body and potentially triggering undesirable immune responses.ResultsThe effect of Lactobacillus plantarum MB452 on tight junction integrity was determined by measuring trans-epithelial electrical resistance (TEER) across Caco-2 cell layers. L. plantarum MB452 caused a dose-dependent TEER increase across Caco-2 cell monolayers compared to control medium. Gene expression was compared in Caco-2 cells untreated or treated with L. plantarum MB452 for 10 hours. Caco-2 cell RNA was hybridised to human oligonucleotide arrays. Data was analysed using linear models and differently expressed genes were examined using pathway analysis tools. Nineteen tight junction-related genes had altered expression levels in response to L. plantarum MB452 (modified-P < 0.05, fold-change > 1.2), including those encoding occludin and its associated plaque proteins that anchor it to the cytoskeleton. L. plantarum MB452 also caused changes in tubulin and proteasome gene expression levels which may be linked to intestinal barrier function. Caco-2 tight junctions were visualised by fluorescent microscopy of immuno-stained occludin, zona occludens (ZO)-1, ZO-2 and cingulin. Caco-2 cells treated with L. plantarum MB452 had higher intensity fluorescence of each of the four tight junction proteins compared to untreated controls.ConclusionsThis research indicates that enhancing the expression of genes involved in tight junction signalling is a possible mechanism by which L. plantarum MB452 improves intestinal barrier function.

Lactobacillus plantarum DSM 2648 is a potential probiotic that enhances intestinal barrier function

The aim of this research was to identify bacterial isolates having the potential to improve intestinal barrier function. Lactobacillus plantarum strains and human oral isolates were screened for their ability to enhance tight junction integrity as measured by the transepithelial electrical resistance (TEER) assay. Eight commercially used probiotics were compared to determine which had the greatest positive effect on TEER, and the best-performing probiotic strain, Lactobacillus rhamnosus HN001, was used as a benchmark to evaluate the isolates. One isolate, L. plantarum DSM 2648, was selected for further study because it increased TEER 135% more than L. rhamnosus HN001. The ability of L. plantarum DSM 2648 to tolerate gastrointestinal conditions and adhere to intestinal cells was determined, and L. plantarum DSM 2648 performed better than L. rhamnosus HN001 in all the assays. Lactobacillus plantarum DSM 2648 was able to reduce the negative effect of Escherichia coli [enteropathogenic E. coli (EPEC)] O127:H6 (E2348/69) on TEER and adherence by as much as 98.75% and 80.18%, respectively, during simultaneous or prior coculture compared with EPEC incubation alone. As yet, the precise mechanism associated with the positive effects exerted by L. plantarum DSM 2648 are unknown, and may influence its use to improve human health and wellness.

The role of cell surface architecture of lactobacilli in host-microbe interactions in the gastrointestinal tract.

Lactobacillus species can exert health promoting effects in the gastrointestinal tract (GIT) through many mechanisms, which include pathogen inhibition, maintenance of microbial balance, immunomodulation, and enhancement of the epithelial barrier function. Different species of the genus Lactobacillus can evoke different responses in the host, and not all strains of the same species can be considered beneficial. Strain variations may be related to diversity of the cell surface architecture of lactobacilli and the bacterias ability to express certain surface components or secrete specific compounds in response to the host environment. Lactobacilli are known to modify their surface structures in response to stress factors such as bile and low pH, and these adaptations may help their survival in the face of harsh environmental conditions encountered in the GIT. In recent years, multiple cell surface-associated molecules have been implicated in the adherence of lactobacilli to the GIT lining, immunomodulation, and protective effects on intestinal epithelial barrier function. Identification of the relevant bacterial ligands and their host receptors is imperative for a better understanding of the mechanisms through which lactobacilli exert their beneficial effects on human health.

Can Nutritional Modulation of Maternal Intestinal Microbiota Influence the Development of the Infant Gastrointestinal Tract

The gastrointestinal microbiota plays an important role in maintaining host health by preventing the colonization of pathogens, fermenting dietary compounds, and maintaining normal mucosal immunity. Particularly in early life, the composition of the microbiota profoundly influences the development and maturation of the gastrointestinal tract (GIT) mucosa, which may affect health in later life. Therefore, strategies to manipulate the microbiota during infancy may prevent the development of some diseases later in adult life. Earlier research suggested that term fetuses are sterile and that the initial bacterial colonization of the newborn GIT occurs only after the baby transits through the birth canal. However, recent studies have demonstrated that the colonization and/or contact of the fetus with the maternal GIT microbiota may start in utero. After vaginal birth, the colonization of the neonate GIT continues through contact with maternal feces and vaginal bacteria, leading to a relatively simple microbial community that is influenced by feeding type (breast vs. formula feeding). Maternal GIT microbiota, vaginal microbiota, and breast milk composition are influenced by maternal diet. Alterations of the maternal GIT microbiota composition via supplementation with probiotics and prebiotics have been shown; however, transfer of these benefits to the offspring remains to be demonstrated. This review focuses on the influence of maternal GIT microbiota during the pre- and postpartum periods on the colonization of the infant GIT. In particular, it examines the manipulation of the maternal GIT microbiota composition through the use of probiotics and/or prebiotics and subsequent consequences for the health of the offspring.

The effects of dietary curcumin and rutin on colonic inflammation and gene expression in multidrug resistance gene-deficient (mdr1a-/-) mice, a model of inflammatory bowel diseases.

Damage of the intestinal epithelial barrier by xenobiotics or reactive oxygen species and a dysregulated immune response are both factors involved in the pathogenesis of inflammatory bowel diseases (IBD). Curcumin and rutin are polyphenolic compounds known to have antioxidant and anti-inflammatory activities, but their mechanism(s) of action are yet to be fully elucidated. Multidrug resistance gene-deficient (mdr1a-/- ) mice spontaneously develop intestinal inflammation, predominantly in the colon, with pathology similar to IBD, so this mouse model is relevant for studying diet-gene interactions and potential effects of foods on remission or development of IBD. The present study tested whether the addition of curcumin or rutin to the diet would alleviate colonic inflammation in mdr1a-/- mice. Using whole-genome microarrays, the effect of dietary curcumin on gene expression in colon tissue was also investigated. Twelve mice were randomly assigned to each of three diets (control (AIN-76A), control +0.2% curcumin or control +0.1% rutin) and monitored from the age of 7 to 24 weeks. Curcumin, but not rutin, significantly reduced histological signs of colonic inflammation in mdr1a-/- mice. Microarray and pathway analyses suggested that the effect of dietary curcumin on colon inflammation could be via an up-regulation of xenobiotic metabolism and a down-regulation of pro-inflammatory pathways, probably mediated by pregnane X receptor (Pxr) and peroxisome proliferator-activated receptor alpha (Ppara) activation of retinoid X receptor (Rxr). These results indicate the potential of global gene expression and pathway analyses to study and better understand the effect of foods in modulating colonic inflammation.

Genome-wide analysis of dietary eicosapentaenoic acid- and oleic acid-induced modulation of colon inflammation in interleukin-10 gene-deficient mice.

Background/Aims: Dietary n–3 polyunsaturated fatty acids can reduce inflammation via a range of mechanisms. This study tested the effect of dietary eicosapentaenoic acid (EPA) on intestinal inflammation using interleukin-10 gene-deficient (Il10–/–) mice. Methods: At 35 days of age, 12 weaned Il10–/– and 12 C57 mice were randomly assigned to one of two modified AIN-76A diets, supplemented with 3.7% purified ethyl esters of either EPA (n–3) or oleic acid (OA, control). To identify genes relevant to colon inflammation, transcription profiling (microarrays and qRT-PCR) and bioinformatic analyses were used. Results: In this study, dietary EPA reversed the decrease in colon fatty acid β-oxidation gene expression observed in OA-fed Il10–/– compared to C57 mice. Il10–/– mice fed the OA diet showed decreased expression of antioxidant enzyme genes, as well as those involved in detoxification of xenobiotics, compared to C57 mice on the same diet. In contrast, dietary EPA increased the expression of these genes in Il10–/– mice. Conclusions: These data indicate that dietary EPA-induced endogenous lipid oxidation which might have a potential anti-inflammatory effect on colon tissue. This is supported by the activation of the Ppara gene that regulates the expression of pro-inflammatory and immunomodulatory genes and proteins.

Metabolomic analysis identifies inflammatory and noninflammatory metabolic effects of genetic modification in a mouse model of Crohn's disease.

Interleukin-10 is an immunosuppressive cytokine involved in the regulation of gastrointestinal mucosal immunity toward intestinal microbiota. Interleukin-10-deficient (IL10(-/-)) mice develop Crohns disease-like colitis unless raised in germ-free conditions. Previous gas chromatography-mass spectrometry (GC-MS) metabolomic analysis revealed urinary metabolite differences between IL10(-/-) and wildtype C57BL/6 mice. To determine which of these differences were specifically associated with intestinal inflammation arising from IL10-deficiency, urine samples from IL10(-/-) and wildtype mice, housed in either conventional or specific pathogen-free conditions, were subjected to GC-MS metabolomic analysis. Fifteen metabolite differences, including fucose, xanthurenic acid, and 5-aminovaleric acid, were associated with intestinal inflammation. Elevated urinary levels of xanthurenic acid in IL10(-/-) mice were attributed to increased production of kynurenine metabolites that may induce T-cell tolerance toward intestinal microbiota. Liquid chromatography-mass spectrometry analysis confirmed that plasma levels of kynurenine and 3-hydroxykynurenine were elevated in IL10(-/-) mice. Eleven metabolite differences, including glutaric acid, 2-hydroxyglutaric acid, and 2-hydroxyadipic acid, were unaffected by the severity of inflammation. These metabolite differences may be associated with residual genes from the embryonic stem cells of the 129P2 mouse strain that were used to create the IL10(-/-) mouse, or may indicate novel functions of IL10 unrelated to inflammation.

Changes in colon gene expression associated with increased colon inflammation in interleukin-10 gene-deficient mice inoculated with Enterococcus species.

BackgroundInappropriate responses to normal intestinal bacteria may be involved in the development of Inflammatory Bowel Diseases (IBD, e.g. Crohns Disease (CD), Ulcerative Colitis (UC)) and variations in the host genome may mediate this process. IL-10 gene-deficient (Il10-/-) mice develop CD-like colitis mainly in the colon, in part due to inappropriate responses to normal intestinal bacteria including Enterococcus strains, and have therefore been used as an animal model of CD. Comprehensive characterization of changes in cecum gene expression levels associated with inflammation in the Il10-/-mouse model has recently been reported. Our aim was to characterize changes in colonic gene expression levels in Il10-/-and C57BL/6J (C57; control) mice resulting from oral bacterial inoculation with 12 Enterococcus faecalis and faecium (EF) strains isolated from calves or poultry, complex intestinal flora (CIF) collected from healthy control mice, or a mixture of the two (EF·CIF). We investigated two hypotheses: (1) that oral inoculation of Il10-/-mice would result in greater and more consistent intestinal inflammation than that observed in Il10-/-mice not receiving this inoculation, and (2) that this inflammation would be associated with changes in colon gene expression levels similar to those previously observed in human studies, and these mice would therefore be an appropriate model for human CD.ResultsAt 12 weeks of age, total RNA extracted from intact colon was hybridized to Agilent 44 k mouse arrays. Differentially expressed genes were identified using linear models for microarray analysis (Bioconductor), and these genes were clustered using GeneSpring GX and Ingenuity Pathways Analysis software. Intestinal inflammation was increased in Il10-/-mice as a result of inoculation, with the strongest effect being in the EF and EF·CIF groups. Genes differentially expressed in Il10-/-mice as a result of EF or EF·CIF inoculation were associated with the following pathways: inflammatory disease (111 genes differentially expressed), immune response (209 genes), antigen presentation (11 genes, particularly major histocompatability complex Class II), fatty acid metabolism (30 genes) and detoxification (31 genes).ConclusionsOur results suggest that colonic inflammation in Il10-/-mice inoculated with solutions containing Enterococcus strains is associated with gene expression changes similar to those of human IBD, specifically CD, and that with the EF·CIF inoculum in particular this is an appropriate model to investigate food-gene interactions relevant to human CD.

Global DNA methylation measurement by HPLC using low amounts of DNA.

Epigenetic changes in chromatin structure can influence gene expression without affecting the DNA sequence. The most commonly studied epigenetic modification, DNA methylation, has been implicated in normal tissue development and disease progression, and can be influenced by diet and other environmental factors. Current HPLC methods of determining DNA methylation may require relatively large amounts of DNA (50 μg); as many tissues have low DNA yields, this can be hard to achieve. We isolated DNA from mouse colon and liver in a study investigating post-natal supplementation with selenium and folic acid. After optimizing the methods to account for lower initial DNA amounts, we digested 3 μg of DNA to deoxynucleotide monophosphates, then purified and quantified it. Samples were analyzed by reversed-phase HPLC to determine global DNA methylation levels using commercial nucleotide standards. The HPLC column was cooled to 6(C (reducing run time), and detection was at 280 nm (UV). We showed that methylated cytosine can be accurately and reproducibly measured in as little as 3 μg of DNA using this HPLC analysis method (within-assay CV <2%). We also used this method to detect reduced DNA methylation in liver (P = 0.009) in response to post-natal supplementation with selenium and folate.