There is some evidence that zinc oxide (ZnO) protects against intestinal diseases. However, despite the suggestions that ZnO may have an antibacterial effect, the mechanisms of this protective effect have not yet been elucidated. We investigated the potential benefits of ZnO in protecting intestinal cells from damage induced by enterotoxigenic Escherichia coli (ETEC, strain K88) and the related mechanisms, using human Caco-2 enterocytes. Cell permeability, measured as transepithelial electrical resistance (TEER), was unaffected by 0.01 and 1 mmol/L ZnO treatments and moderately increased by 5 mmol/L ZnO, compared with untreated cells. Transfer of ¹⁴C-inulin was slightly increased by 5 mmol/L ZnO compared with untreated cells; transfer was unaffected by lower concentrations. The TEER and ¹⁴C-inulin transfer were lower in ETEC-infected cells than in uninfected cells. Treatment of ETEC exposure with 0.2 mmol/L ZnO prevented disruption of membrane integrity. The ETEC was able to adhere to enterocytes and, to some extent, invade the cells. The ZnO treatment reduced bacterial adhesion and blocked bacterial invasion. The ETEC infection upregulated the expression of the inflammatory cytokines interleukin-8, growth-related oncogene-α and tumor necrosis factor-α, and reduced that of the anti-inflammatory cytokine transforming growth factor-β, compared with uninfected cells. The addition of 0.2 or 1 mmol/L ZnO counteracted the alteration of cytokine mRNA levels caused by ETEC. The protective effects of ZnO were not due to any antibacterial activity, because the viability of ETEC grown in a medium containing ZnO was unaffected. In conclusion, ZnO may protect intestinal cells from ETEC infection by inhibiting the adhesion and internalization of bacteria, preventing the increase of tight junction permeability and modulating cytokine gene expression.

Probiotic bacteria may provide protection against intestinal damage induced by pathogens, but the underlying mechanisms are still largely unknown. We investigated whetherBifidobacterium animalisMB5 andLactobacillus rhamnosusGG (LGG) protected intestinal Caco-2 cells from the inflammation-associated response induced by enterotoxigenicEscherichia coli(ETEC) K88, by inhibiting pathogen attachment to the cells, which is the first step of ETEC pathogenicity, and regulating neutrophil recruitment, a crucial component of inflammation. A partial reduction of ETEC adhesion was exerted by probiotics and their culture supernatant fractions either undigested or digested with proteases. ETEC viability was unaffected by the presence ofB. animalis, LGG or their supernatant fractions in the culture medium, indicating an absence of probiotic bactericidal activity. Probiotics and their supernatant fractions, either undigested or digested with proteases, strongly inhibited the neutrophil transmigration caused by ETEC. BothB. animalisand LGG counteracted the pathogen-induced up regulation of IL-8, growth-related oncogene-α and epithelial neutrophil-activating peptide-78 gene expression, which are chemokines essential for neutrophil migration. Moreover, the probiotics prevented the ETEC-induced increased expression of IL-1β and TNF-α and decrease of transforming growth factor-α, which are regulators ofchemokine expression. These results indicate thatB. animalisMB5 and LGG protect intestinal cells from the inflammation-associated response caused by ETEC K88 by partly reducing pathogen adhesion and by counteracting neutrophil migration, probably through the regulation of chemokine and cytokine expression.

Lactobacilli have a potential to overcome intestinal disorders; however, the exact mode of action is still largely unknown. In this study, we have used the intestinal porcine intestinal IPEC-1 epithelial cells as a model to investigate a possible protective activity of a new Lactobacillus species, the L. sobrius DSM 16698^T, against intestinal injury induced by enterotoxigenic Escherichia coli (ETEC) K88 infection and the underlying mechanisms. Treatment of infected cells with L. sobrius strongly reduced the pathogen adhesion. L. sobrius was also able to prevent the ETEC-induced membrane damage by inhibiting delocalization of zonula occludens (ZO)-1, reduction of occludin amount, rearrangement of F-actin, and dephosphorylation of occludin caused by ETEC. RT-PCR and ELISA experiments showed that L. sobrius counteracted the ETEC-induced increase of IL-8 and upregulated the IL-10 expression. The involvement of IL-8 in the deleterious effects of ETEC was proven by neutralization of IL-8 with a specific antibody. A crucial role of IL-10 was indicated by blockage of IL-10 production with neutralizing anti-IL-10 antibody that fully abrogated the L. sobrius protection. L. sobrius was also able to inhibit the internalization of ETEC, which was likely favored by the leaking barrier. The protective effects were not found with L. amylovorus DSM 20531^T treatment, a strain derived from cattle waste but phylogenetically closely related to L. sobrius. Together, the data indicate that L. sobrius exerts protection against the harmful effects of ETEC by different mechanisms, including pathogen adhesion inhibition and maintenance of membrane barrier integrity through IL-10 regulation.

Fumonisin B₁ (FB₁) is a mycotoxin produced by Fusarium verticillioides (formerly F. moniliforme), a fungus that commonly contaminates maize. FB₁ causes toxicological effects in laboratory and domestic animals including pigs. Because the gastrointestinal tract represents the first barrier met by exogenous food compounds, the purpose of this study was to investigate the effects of FB₁ on IPEC-1, a porcine intestinal epithelial cell line. We first verified that low concentrations of FB₁ did not exert any cytotoxic effect on IPEC-1. Indeed, significant LDH release was only observed for FB₁ concentrations greater than 50 and 700 μM on proliferating and nonproliferating cells, respectively. We then demonstrated that FB₁ inhibits proliferation of IPEC-1. Fluorescence-activated cell sorting (FACS) analysis of the cell cycle indicated that FB₁ blocks the proliferation of intestinal cells in the G0/G1 phase. Similar results were obtained with LLC-PK1, a renal porcine epithelial cell line, which is considered to be a good model for studying FB₁ in vitro effects. We have also assessed the effects of FB₁ on the integrity of the barrier formed by the intestinal epithelium. We demonstrated that FB₁ decreases the transepithelial electrical resistance (TEER) of IPEC-1 in a time- and dose-dependent manner. This effect was only noticed after a long exposure (8–12 days of treatment). FB₁ induced the TEER decrease independently of the cell differentiation stage, and this effect was partially reversible. Taken together, our data indicate that FB₁ alters the proliferation and the barrier function of intestinal cells. These results may have implications for humans and animals consuming FB₁-contaminated food or feed.

Inflammation derived from pathogen infection involves the activation of toll-like receptor (TLR) signaling. Despite the established immunomodulatory activities of probiotics, studies relating the ability of such bacteria to inhibit the TLR signaling pathways are limited or controversial. In a previous study we showed that Lactobacillus amylovorus DSM 16698^T, a novel lactobacillus isolated from unweaned pigs, protects the intestinal cells from enterotoxigenic Escherichia coli (ETEC) K88 infection through cytokine regulation. In the present study we investigated whether the ability of L. amylovorus to counteract the inflammatory status triggered by ETEC in intestine is elicited through inhibition of the TLR4 signaling pathway. We used the human intestinal Caco-2/TC7 cells and intestinal explants isolated from 5 week-old crossbreed Pietrain/Duroc/Large-White piglets, treated with ETEC, L. amylovorus or L. amylovorus cell free supernatant, either alone or simultaneously with ETEC. Western blot analysis showed that L. amylovorus and its cell free supernatant suppress the activation of the different steps of TLR4 signaling in Caco-2/TC7 cells and pig explants, by inhibiting the ETEC induced increase in the level of TLR4 and MyD88, the phosphorylation of the IKKα, IKKβ, IκBα and NF-κB subunit p65, as well as the over-production of inflammatory cytokines IL-8 and IL-1β. The immunofluorescence analysis confirms the lack of phospho-p65 translocation into the nucleus. These anti-inflammatory effects are achieved through modulation of the negative regulators Tollip and IRAK-M. We also found that L. amylovorus blocks the up-regulation of the extracellular heat shock protein (Hsp)72 and Hsp90, that are critical for TLR4 function. By using anti-TLR2 antibody, we demonstrate that TLR2 is required for the suppression of TLR4 signaling activation. These results may contribute to develop therapeutic interventions using L. amylovorus in intestinal disorders of piglets and humans.

Background. Probiotics may protect against inflammatory bowel disease through regulation of lamina propria lymphocytes (LPLs) function. Data are lacking