Christoph Cichon

Molecular mechanisms underlying the probiotic effects of Escherichia coli Nissle 1917 involve ZO-2 and PKCzeta redistribution resulting in tight junction and epithelial barrier repair.

The probiotic Escherichia coli strain Nissle 1917 (EcN) has been used for decades in human medicine in Central Europe for the treatment and prevention of intestinal disorders and diseases. However, the molecular mechanisms underlying its beneficial effects are only partially understood. To identify molecular responses induced by EcN that might contribute to its probiotic properties polarized T84 cells were investigated employing DNA microarrays, quantitative RT‐PCR, Western blotting, immunofluorescence and specific protein kinase C (PKC) inhibitors. Polarized T84 epithelial cell monolayers were used as a model to monitor barrier disruption by infection with the enteropathogenic E. coli (EPEC) strain E2348/69. Co‐incubation of EPEC with EcN or addition of EcN following EPEC infection abolished barrier disruption and, moreover, restored barrier integrity as monitored by transepithelial resistance. DNA‐microarray analysis of T84 cells incubated with EcN identified 300+ genes exhibiting altered expression. EcN altered the expression, distribution of zonula occludens‐2 (ZO‐2) protein and of distinct PKC isotypes. ZO‐2 expression was enhanced in parallel to its redistribution towards the cell boundaries. This study provides evidence that EcN induces an overriding signalling effect leading to restoration of a disrupted epithelial barrier. This is transmitted via silencing of PKCζ and the redistribution of ZO‐2. We suggest that these properties contribute to the reported efficacy in the treatment of inflammatory bowel diseases and in part rationalize the probiotic nature of EcN.

Differential targeting of the E-Cadherin/β-Catenin complex by gram-positive probiotic lactobacilli improves epithelial barrier function.

ABSTRACT The intestinal ecosystem is balanced by dynamic interactions between resident and incoming microbes, the gastrointestinal barrier, and the mucosal immune system. However, in the context of inflammatory bowel diseases (IBD), where the integrity of the gastrointestinal barrier is compromised, resident microbes contribute to the development and perpetuation of inflammation and disease. Probiotic bacteria have been shown to exert beneficial effects, e.g., enhancing epithelial barrier integrity. However, the mechanisms underlying these beneficial effects are only poorly understood. Here, we comparatively investigated the effects of four probiotic lactobacilli, namely, Lactobacillus acidophilus, L. fermentum, L. gasseri, and L. rhamnosus, in a T84 cell epithelial barrier model. Results of DNA microarray experiments indicating that lactobacilli modulate the regulation of genes encoding in particular adherence junction proteins such as E-cadherin and β-catenin were confirmed by quantitative reverse transcription-PCR (qRT-PCR). Furthermore, we show that epithelial barrier function is modulated by Gram-positive probiotic lactobacilli via their effect on adherence junction protein expression and complex formation. In addition, incubation with lactobacilli differentially influences the phosphorylation of adherence junction proteins and the abundance of protein kinase C (PKC) isoforms such as PKCδ that thereby positively modulates epithelial barrier function. Further insight into the underlying molecular mechanisms triggered by these probiotics might also foster the development of novel strategies for the treatment of gastrointestinal diseases (e.g., IBD).

MicroRNAs regulate tight junction proteins and modulate epithelial/endothelial barrier functions

Tightly controlled epithelial and endothelial barriers are a prerequisite for life as these barriers separate multicellular organisms from their environment and serve as first lines of defense. Barriers between neighboring epithelial cells are formed by multiple intercellular junctions including the ‘apical junctional complex—AJC’ with tight junctions (TJ), adherens junctions (AJ), and desmosomes. TJ consist of tetraspan transmembrane proteins like occludin, various claudins that directly control paracellular permeability, and the ‘Junctional Adhesion Molecules’ (JAMs). For establishing tight barriers TJ are essential but at the same time have to allow also selective permeability. For this, TJ need to be tightly regulated and controlled. This is organized by a variety of adaptor molecules, i.e., protein kinases, phosphatases and GTPases, which in turn are regulated and fine-tuned involving microRNAs (miRNAs). In this review we summarize available data on the role and targeting of miRNAs in the maintenance of epithelial and/or endothelial barriers.

Association of Shiga toxin glycosphingolipid receptors with membrane microdomains of toxin-sensitive lymphoid and myeloid cells

Glycosphingolipids (GSLs) of the globo-series constitute specific receptors for Shiga toxins (Stxs) released by certain types of pathogenic Escherichia coli strains. Stx-loaded leukocytes may act as transporter cells in the blood and transfer the toxin to endothelial target cells. Therefore, we performed a thorough investigation on the expression of globo-series GSLs in serum-free cultivated Raji and Jurkat cells, representing B- and T-lymphocyte descendants, respectively, as well as THP-1 and HL-60 cells of the monocyte and granulocyte lineage, respectively. The presence of Stx-receptors in GSL preparations of Raji and THP-1 cells and the absence in Jurkat and HL-60 cells revealed high compliance of solid-phase immunodetection assays with the expression profiles of receptor-related glycosyltransferases, performed by qRT-PCR analysis, and Stx2-caused cellular damage. Canonical microdomain association of Stx GSL receptors, sphingomyelin, and cholesterol in membranes of Raji and THP-1 cells was assessed by comparative analysis of detergent-resistant membrane (DRM) and nonDRM fractions obtained by density gradient centrifugation and showed high correlation based on nonparametric statistical analysis. Our comprehensive study on the expression of Stx-receptors and their subcellular distribution provides the basis for exploring the functional role of lipid raft-associated Stx-receptors in cells of leukocyte origin.

Identification of specific miRNAs targeting proteins of the apical junctional complex that simulate the probiotic effect of E. coli Nissle 1917 on T84 epithelial cells.

In the intestine, dysregulation of miRNA is associated with inflammation, disruption of the gastrointestinal barrier, and the onset of gastrointestinal disorders. This study identifies miRNAs involved in the maintenance of intercellular junctions and barrier integrity. For the functional identification of barrier affecting miRNAs, we took advantage of the barrier-enforcing effects of the probiotic bacterium Escherichia coli Nissle 1917 (EcN) which can be monitored by enhanced transepithelial resistance (TER). miRNA-profiling of T84 monolayers prior and after co-incubation with EcN revealed for the first time differentially regulated miRNAs (miR-203, miR-483-3p, miR-595) targeting tight junction (TJ) proteins. Using real-time PCR, Western blotting and specific miRNA mimics, we showed that these miRNAs are involved in the regulation of barrier function by modulating the expression of regulatory and structural components of tight junctional complexes. Furthermore, specific inhibitors directed at these miRNA abrogated the disturbance of tight junctions induced by enteropathogenic E. coli (EPEC). The half-maximal inhibitory concentration (IC(50)) was determined to 340 nM by monitoring inhibitor kinetics. In summary, we conclude that specific miRNAs effect regulatory as well as structural proteins of the junctional complex which in turn are involved in the barrier enhancing effect of EcN. Hence, we suggest that the application of miRNAs might be refined and further developed as a novel supportive strategy for the treatment of gastrointestinal disorders.

Interleukin-8, CXCL1, and MicroRNA miR-146a Responses to Probiotic Escherichia coli Nissle 1917 and Enteropathogenic E. coli in Human Intestinal Epithelial T84 and Monocytic THP-1 Cells after Apical or Basolateral Infection

ABSTRACT Bacterium-host interactions in the gut proceed via directly contacted epithelial cells, the hosts immune system, and a plethora of bacterial factors. Here we characterized and compared exemplary cytokine and microRNA (miRNA) responses of human epithelial and THP-1 cells toward the prototype enteropathogenic Escherichia coli (EPEC) strain E2348/69 (O127:H6) and the probiotic strain Escherichia coli Nissle 1917 (EcN) (O6:K5:H1). Human T84 and THP-1 cells were used as cell culture-based model systems for epithelial and monocytic cells. Polarized T84 monolayers were infected apically or basolaterally. Bacterial challenges from the basolateral side resulted in more pronounced cytokine and miRNA responses than those observed for apical side infections. Interestingly, the probiotic EcN also caused a pronounced transcriptional increase of proinflammatory CXCL1 and interleukin-8 (IL-8) levels when human T84 epithelial cells were infected from the basolateral side. miR-146a, which is known to regulate adaptor molecules in Toll-like receptor (TLR)/NF-κB signaling, was found to be differentially regulated in THP-1 cells between probiotic and pathogenic bacteria. To assess the roles of flagella and flagellin, we employed several flagellin mutants of EcN. EcN flagellin mutants induced reduced IL-8 as well as CXCL1 responses in T84 cells, suggesting that flagellin is an inducer of this cytokine response. Following infection with an EPEC type 3 secretion system (T3SS) mutant, we observed increased IL-8 and CXCL1 transcription in T84 and THP-1 cells compared to that in wild-type EPEC. This study emphasizes the differential induction of miR-146a by pathogenic and probiotic E. coli strains in epithelial and immune cells as well as a loss of probiotic properties in EcN interacting with cells from the basolateral side.

MicroRNA-320a Strengthens Intestinal Barrier Function and Follows the Course of Experimental Colitis.

Background:Inflammatory bowel disease is a chronic-remittent disorder with the risk of disabling complications due to uncontrolled inflammation. Accurate biomarkers are needed to noninvasively monitor the disease course to tailor therapy. We evaluated the potential of the specific microRNA (miR)-320a to monitor disease activity in experimental colitis or patients with Crohns disease and investigated its functional role in intestinal epithelial barrier formation. Methods:The impact of miR-320a on intestinal barrier function was tested in vitro in T84 epithelial cells by transepithelial resistance measurement and quantitative real-time polymerase chain reaction analysis on inflammatory and microbial stimulation. Experimental colitis was studied in dextran sodium sulfate colitis, T-cell transfer colitis, and IL-10-/- mice. Disease course was monitored by body weight measurement, colonoscopy, and histological examination. MiR-320a expression during inflammation was assessed in T84 cells, murine blood, and colonic tissue and in peripheral blood from patients with Crohns disease with active or quiescent disease. Results:MiR-320a transfection of T84 cells reinforced barrier integrity reflected by increased transepithelial resistance (P < 0.01) and inhibited barrier-destructive enteropathogenic Escherichia coli effects resulting in increased tight junction protein JAM-A expression (P = 0.02) and decrease of barrier integrity–destabilizing miR-320a target PPP2R5B (P < 0.001). Tumor necrosis factor-&agr; and interleukin-1&bgr; stimulation increased a miR-320a epxression in T84 cells. MiR-320a level was increased in blood samples from colitic mice and patients with Crohns disease showing a strong correlation with disease activity (r2 = 0.67). Conclusions:MiR-320a strengthens intestinal barrier function in vitro and has the potential to monitor disease activity of colitic mice. Future studies are needed to further evaluate the potential of miR-320a in patients with inflammatory bowel disease.