The intestinal epithelium forms a protective barrier against luminal contents and the external environment, mediated via intercellular tight junctions (TJs). The TJ can be disrupted via cell signalling induced by either enteric pathogens or pro-inflammatory cytokines, thereby contributing to various intestinal disorders ranging from acute infectious diarrhoea to chronic inflammatory bowel diseases. Probiotics, such as Lactobacillus rhamnosus GG (LGG), are reported to confer beneficial effects on epithelial cells, including antagonizing infections and reducing overt proinflammatory responses, but the underlying mechanisms of these observed effects require further characterization. We hypothesized that probiotics preserve barrier function by interfering with proinflammatory cytokine signalling. Caco-2bbe cells were seeded into Transwells to attain polarized monolayers with intercellular TJs. Monolayers were inoculated apically with the probiotic LGG 3 h prior to the addition of IFN-c (100 ng ml "1 ) to the basolateral medium overnight. The monolayers were then placed in fresh basal medium±TNF-a (10 ng ml "1) and transepithelial electrical resistance (TER) measurements were taken over the time-course of TNF-a stimulation. To complement the TER findings, cells were processed for zona occludens-1 (ZO-1) immunofluorescence staining. As a measure of TNF-a downstream signalling, cells were immunofluorescently stained for NF-kB p65 subunit and CXCL-8 mRNA was quantified by qRT-PCR. Basal cell culture medium was collected after overnight TNF-a stimulation to measure secreted chemokines, including CXCL-8 (interleukin-8) and . Following LGG inoculation, IFN-c priming and 24 h TNF-a stimulation, epithelial cells maintained TER and ZO-1 distribution.LGG diminished the nuclear translocation of p65, demonstrated by both immunofluorescence and CXCL-8 mRNA expression. CXCL-8 and CCL-11 protein levels were decreased in LGG-inoculated, cytokine-challenged cells. These findings indicate that LGG alleviates the effects of pro-inflammatory cytokines on epithelial barrier integrity and inflammation, mediated, at least in part, through inhibition of NF-kB signalling. INTRODUCTIONThe intestinal epithelium forms a protective barrier against luminal contents, such as microbes and dietary food antigens, present in the external environment. Barrier function is mediated through apical junction complexes, which consist of paracellular proteins integrated into tight junctions (TJs) (Van Itallie & Anderson, 2006). Abnormalities of intercellular TJs contribute to a variety of intestinal disorders, including acute diarrhoeal illness, gluten-sensitive enteropathy (coeliac disease) and chronic inflammatory bowel disease (McGuckin et al., 2009). The challenging of intact polarized epithelia with either proinflammatory cytokines or pathogenic bacteria dismantles the TJ protein structure and thereby disrupts barrier function (Donato et al., 2008; Wang et al., 2005Wang et al., , 2006 Zareie et al., 2005).We have shown that probiotic lactobacilli antago...
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 intimately attaches to intestinal epithelial monolayers and produces attaching and effacing (A/E) lesions. In addition, EHEC infection causes disruptions of intercellular tight junctions, leading to clinical sequelae that include acute diarrhea, hemorrhagic colitis, and the hemolytic-uremic syndrome. Current therapy remains supportive since antibiotic therapy increases the risk of systemic complications. This study focused on the potential therapeutic effect of an alternative form of therapy, probiotic Lactobacillus rhamnosus strain GG, to attenuate EHEC-induced changes in paracellular permeability in polarized MDCK-I and T84 epithelial cell monolayers. Changes in epithelial cell morphology, electrical resistance, dextran permeability, and distribution and expression of claudin-1 and ZO-1 were assessed using phase-contrast, immunofluorescence, and transmission electron microscopy and macromolecular flux. This study demonstrated that pretreatment of polarized MDCK-I and T84 cells with the probiotic L. rhamnosus GG reduced morphological changes and diminished the number of A/E lesions induced in response to EHEC O157:H7 infection. With probiotic pretreatment there was corresponding attenuation of the EHEC-induced drop in electrical resistance and the increase in barrier permeability assays. In addition, L. rhamnosus GG protected epithelial monolayers against EHEC-induced redistribution of the claudin-1 and ZO-1 tight junction proteins. In contrast to the effects seen with the live probiotic, heat-inactivated L. rhamnosus GG had no effect on EHEC binding and A/E lesion formation or on disruption of the barrier function. Collectively, these findings provide in vitro evidence that treatment with the probiotic L. rhamnosus strain GG could prove to be an effective management treatment for preventing injury of the epithelial cell barrier induced by A/E bacterial enteropathogens.
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