Differing roles of protein kinase C-zeta in disruption of tight junction barrier by enteropathogenic and enterohemorrhagic Escherichia coli

BACKGROUND & AIMS: Enteropathogenic Escherichia coli and enterohemorrhagic E. coli harbor highly homologous pathogenicity islands yet show key differences in their mechanisms of action. Both disrupt host intestinal epithelial tight junctions, but the effects of enteropathogenic E. coli are more profound than those of enterohemorrhagic E. coli. The basis for this is not understood. The atypical protein kinase C isoform, protein kinase C-zeta, associates with and regulates the tight junction complex. The aim of this study was to compare the role of protein kinase C-zeta in the disruption of tight junctions after infection with enteropathogenic E. coli and enterohemorrhagic E. coli. METHODS: Model intestinal epithelial monolayers infected by enteropathogenic E. coli or enterohemorrhagic E. coli were used for these studies. RESULTS: Neither bisindolylmaleimide nor G?6976, which block several protein kinase C isoforms but not protein kinase C-zeta, protected against the decrease in transepithelial electrical resistance after enteropathogenic E. coli infection. Rottlerin at concentrations that block novel and atypical isoforms, including protein kinase C-zeta, significantly attenuated the decrease in transepithelial electrical resistance. The specific inhibitory peptide, myristoylated protein kinase C-zeta pseudosubstrate, also significantly decreased the enteropathogenic E. coli -associated decrease in transepithelial electrical resistance and redistribution of tight junction proteins. In contrast to enteropathogenic E. coli, the level of protein kinase C-zeta enzyme activity stimulated by enterohemorrhagic E. coli was transient and minor, and protein kinase C-zeta inhibition had no effect on the decrease in transepithelial electrical resistance or the redistribution of occludin. CONCLUSIONS: The differential regulation of protein kinase C-zeta by enteropathogenic E. coli and enterohemorrhagic E. coli may in part explain the less profound effect of the latter on the barrier function of tight junctions.     

In vitro evaluation of intestinal epithelial TLR activation in preventing food allergic responses

Alterations in the gut microbiota composition are associated with food allergy. Toll-like receptors (TLRs) respond to microbial stimuli. We studied the effects of the ligation of TLRs on intestinal epithelial cells (IECs) in preventing an allergic effector response. IEC monolayers (T84 cells) were co-cultured with CD3/28-activated PBMCs from healthy controls or atopic patients and simultaneously apically exposed to TLR2, TLR4 or TLR9 ligands. The barrier integrity of T84 cell monolayers was significantly reduced upon co-culture with PBMCs of food allergic subjects compared to healthy subjects. Apical exposure of IECs to a TLR9 ligand prevented PBMC-induced epithelial barrier disruption. Using PBMCs from food allergic subjects, apical TLR9 activation on IECs increased the IFN-γ/IL-13 and IL-10/IL-13 ratio, while suppressing pro-inflammatory IL-6, IL-8 and TNF-α production in an IEC-dependent manner. Hence, the activation of apical TLR9 on IECs, potentially by microbiota-derived signals, may play an important role in the prevention of allergic inflammation.     

Mast cell corticotropin-releasing factor subtype 2 suppresses mast cell degranulation and limits the severity of anaphylaxis and stress-induced intestinal permeability

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Toxicity of gold-nanoparticles: Synergistic effects of shape and surface functionalization on micromotility of epithelial cells

Abstract

Nanoparticle exposure is monitored by a combination of two label-free and non-invasive biosensor devices which detect cellular shape and viscoelasticity (quartz crystal microbalance), cell motility and the dynamics of epithelial cell-cell contacts (electric cell-substrate impedance sensing). With these tools we have studied the impact of nanoparticle shape on cellular physiology. Gold (Au) nanoparticles coated with CTAB were synthesized and studied in two distinct shapes: Spheres with a diameter of (43 ± 4) nm and rods with a size of (38 ± 7) nm × (17 ± 3) nm. Dose-response experiments were accompanied by conventional cytotoxicity tests as well as fluorescence and dark-field microscopy to visualize the intracellular particle distribution. We found that spherical gold nanoparticles with identical surface functionalization are generally more toxic and more efficiently ingested than rod-shaped particles. We largely attribute the higher toxicity of CTAB-coated spheres as compared to rod-shaped particles to a higher release of toxic CTAB upon intracellular aggregation.     

Effect of Hydrogen Peroxide on Interleukin-8 Synthesis and Death of Caco-2 Cells

Intestinal epithelial cells can secrete interleukin-8 (IL-8), among other substances in response to different stimuli, which plays an important role in mucosal immune response. Above a certain concentration range, hydrogen peroxide causes cell death by necrosis or apoptosis. We investigated the time- and dose-dependent induction of IL-8 by hydrogen peroxide in the human colon adenocarcinoma cell line Caco-2. In addition, the changes of transepithelial electrical resistance and cell death induction in response to hydrogen peroxide were studied. Nonfilter-grown and filter-grown Caco-2 cells were employed in our experiments. Interleukin-8 synthesis was measured by ELISA. Necrosis was determined by DAPI staining of cells, apoptosis by measuring caspase-3 enzyme activity or annexin V staining. In nonfilter-grown Caco-2 cells, 1 mM of hydrogen peroxide induced the highest level of IL-8 production 24 hr after treatment. In filter-grown Caco-2 cells, IL-8 was produced only on the apical side in response to 1 mM of hydrogen peroxide. This level was 10-fold lower than that measured in nonfilter-grown Caco-2 cells 24 hr after the treatment. In filter-grown Caco-2 cells 10 mM hydrogen peroxide induced the highest IL-8 level on the apical as well as basolateral side. Transepithelial electrical resistance decreased markedly upon application of 40 mM hydrogen peroxide. Late effect of hydrogen peroxide was observed in nonfilter-grown Caco-2 cells, as 1 mM hydrogen peroxide caused necrosis after 24 hr while early-necrosis induction occurred in filter-grown cells exposed to 40 mM of hydrogen peroxide after 1 hr. Filter-grown Caco-2 cells were less sensitive to hydrogen peroxide than the nonfilter-grown ones.     

Investigations on the effects of alkyl polyglucosides on development and fertility.

Alkyl polyglucosides are non-ionic surfactants which are mainly used in laundry detergents, hard surface cleaners and personal care products. Two in vitro screening test systems were used to investigate the endocrine modulating potential of alkyl polyglucosides. No indications were observed for any estrogenic or anti-estrogenic effects in an MCF-7 E-Screen assay and a reporter gene assay using luciferase-transfected MCF-7 cells. Concentrations exceeding the effect concentration of estradiol by a factor of 1000 did not reveal any indication for an estrogenic activity. Furthermore, in a combination assay investigating the effects of estradiol in the presence of alkyl polyglucoside, no anti-estrogenic potential was observed. In an one-generation screening assay in rats no effects on the fertility were observed up to the highest dose of 1000 mg/kg bw/day alkyl glucoside. Potential embryo-/fetotoxicity and/or teratogenicity was investigated in a segment II study in rats. No effects were noted up to the limit dose of 1000 mg/kg bw/day. All parameters of the treatment groups were comparable with those of the control group. Skeletal and visceral investigations did not detect any treatment-related malformations. For the embryo/fetotoxicity, the teratogenicity and the maternal toxicity a NOAEL of 1000 mg/kg was deduced.     

Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization

Phenol contamination of soil and water has raised concerns among people living near phenol-producing factories and hazardous waste sites containing the chemical. Phenol, particularly in high concentrations, is an irritating and corrosive substance, making mucosal membranes targets of toxicity in humans. However, few data on the effects of phenol after oral exposure exist.We used an in vitro model employing human intestinal epithelial cells (SK-CO15) cultured on permeable supports to examine effects of phenol on epithelial barrier function. We hypothesized that phenol disrupts epithelial barrier by altering tight junction (TJ) protein expression. The dose-response effect of phenol on epithelial barrier function was determined using transepithelial electrical resistance (TER) and FITC-dextran permeability measurements. We studied phenol-induced changes in cell morphology and expression of several tight junction proteins by immunofluorescence and Western blot analysis. Effects on cell viability were assessed by MTT, Trypan blue, propidium iodide and TUNEL staining.Exposure to phenol resulted in decreased TER and increased paracellular flux of FITC-dextran in a dose-dependent manner. Delocalization of claudin-1 and ZO-1 from TJs to cytosol correlated with the observed increase in permeability after phenol treatment. Additionally, the decrease in TER correlated with changes in the distribution of a membrane raft marker, suggesting phenol-mediated effects on membrane fluidity. Such observations were independent of effects of phenol on cell viability as enhanced permeability occurred at doses of phenol that did not cause cell death. Overall, these findings suggest that phenol may affect transiently the lipid bilayer of the cell membrane, thus destabilizing TJ-containing microdomains.     

Role of Tyr306 in the C-terminal fragment of Clostridium perfringens enterotoxin for modulation of tight junction

We previously reported that the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is a novel type of absorption enhancer that interacts with claudin-4 and that Tyr306 of C-CPE plays a role in ability of C-CPE to modulate barrier of tight junctions. In the current study, to investigate effects of Tyr306 on the C-CPE activity, we prepared some C-CPE mutants substituted Tyr306 with Trp (Y306W), Phe (Y306F) and Lys (Y306K). We found that Y306W and Y306F mutants of C-CPE had claudin-4 binding affinities and effects on the barrier function of tight junctions, whereas both of these properties were greatly reduced with the Y306K mutant. Finally, the Y306K but not the Y306F and Y306W mutants had reduced abilities to enhance absorption in rat jejunum. These results indicate that aromatic and hydrophobic properties, not hydrogen bonding potential, of Tyr306 are involved in the interaction of C-CPE with claudin-4 and in the modulation of the tight junction barrier function by C-CPE.     

Engineering antigens for in situ erythrocyte binding induces T-cell deletion

Antigens derived from apoptotic cell debris can drive clonal T-cell deletion or anergy, and antigens chemically coupled ex vivo to apoptotic cell surfaces have been shown correspondingly to induce tolerance on infusion. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered two different antigen constructs to target the antigen to erythrocyte cell surfaces after i.v. injection, one using a conjugate with an erythrocyte-binding peptide and another using a fusion with an antibody fragment, both targeting the erythrocyte-specific cell surface marker glycophorin A. Here, we show that erythrocyte-binding antigen is collected much more efficiently than free antigen by splenic and hepatic immune cell populations and hepatocytes, and that it induces antigen-specific deletional responses in CD4⁺ and CD8⁺ T cells. We further validated T-cell deletion driven by erythrocyte-binding antigens using a transgenic islet β cell-reactive CD4⁺ T-cell adoptive transfer model of autoimmune type 1 diabetes: Treatment with the peptide antigen fused to an erythrocyte-binding antibody fragment completely prevented diabetes onset induced by the activated, autoreactive CD4⁺ T cells. Thus, we report a translatable modular biomolecular approach with which to engineer antigens for targeted binding to erythrocyte cell surfaces to induce antigen-specific CD4⁺ and CD8⁺ T-cell deletion toward exogenous antigens and autoantigens.