Lactobacillus plantarum MB452 enhances the function of the intestinal barrier by increasing the expression levels of genes involved in tight junction formation - PubMed (original) (raw)

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

Rachel C Anderson et al. BMC Microbiol. 2010.

Abstract

Background: Intestinal 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.

Results: The 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.

Conclusions: This 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.

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Figures

Figure 1

Figure 1

Change in trans-epithelial electrical resistance (TEER) across confluent Caco-2 monolayers (5 days old) over time in the presence of different optical densities of L. plantarum MB452. The change in TEER is the percentage change compared to the initial TEER for each monolayer. The values plotted are the means for four monolayers and the error bars show the SEM. OD = the starting optical density of the L. plantarum cultures at 600 nm.

Figure 2

Figure 2

Change in trans-epithelial electrical resistance (TEER) across differentiated Caco-2 monolayers (18 days old) in the presence of L. plantarum MB452 (OD600 nm 0.9). The change in TEER is the percentage change compared to the initial TEER for each monolayer. The values plotted are the means for four monolayers and the error bars show the SEM.

Figure 3

Figure 3

Network of genes involved in tight junction formation that were differentially expressed by Caco-2 cells after being co-cultured with L. plantarum MB452 (OD600 nm 0.9) for 10 hours. Genes are represented as nodes and the biological relationship between two nodes is represented as an edge. All edges are supported by at least one reference from the literature. Red and green colored nodes indicate genes that have increased or decreased expression, respectively, in response to L. plantarum MB452. The colors of the gene names indicate the role the encoded proteins in relation to tight junctions.

Figure 4

Figure 4

Fluorescent microscopy images of immuno-stained tight junction proteins of confluent Caco-2 cells (6 days old) untreated or treated with L. plantarum MB452 (OD600 nm 0.9) for 8 hours. Treatments were carried out in quadruplicate and the images shown are typical. ZO-1: zonula occluden 1; ZO-2 zonula occluden 2; OCLN: occludin; CGN: cingulin.

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