Reduction In Transepithelial Resistance Research Articles

Prevalence and virulence potential of Aeromonas spp. isolated from human diarrheal samples in North East Italy.

Aeromonas spp. are emerging human pathogens causing intestinal and extra-intestinal infections. Since their relevance in Western Europe as gastrointestinal pathogens is not well established, we investigated Aeromonas spp. prevalence in diarrheal fecal samples in an Italian University Hospital and characterized the virulence mechanisms of the isolates. Aeromonas spp. isolated from diarrheic stools using standard culture methods were identified by molecular techniques. Antimicrobial resistance was assessed by the micro broth dilution. Toxins, flagella, and type III secretion system genes were evaluated by polymerase chain reaction. Biofilm was quantified by crystal-violet staining. Interaction with human intestinal epithelial cells (Caco-2) was assessed by quantifying adhesion, interleukin (IL)-8 secretion, and epithelial barrier integrity. Aeromonas spp. represented 20.6% of bacterial pathogens isolated from diarrheic feces, the second most common enteropathogen. A. cavieae constituted 75% of the identified species, showing a relatively low clustering value. About 52% of Aeromonas isolates showed resistance to amikacin, whereas only 7.5% showed multiple drug resistance. Four or more virulence genes were identified in 66.7% of A. cavieae isolates and 100% of A. dakensis. Aeromonas isolates (82.5%) showed moderate or important biofilm-producing ability. Adhesion to Caco-2 cells correlated to fla+ gene, whereas ascV+ and aexU+ strains significantly induced IL-8 release from Caco-2. Aeromonas aer+ strains caused ZO1 and occluding redistribution and a significative reduction in trans-epithelial resistance. Aeromonas spp. emerge as relevant human intestinal pathogens with a disparate arsenal of pathogenicity factors causing diarrhea through different mechanisms. IMPORTANCE In this work, we demonstrate the epidemiologic relevance of the Aeromonas genus as the cause of infective diarrhea in North East Italy, both in children and adult subjects, with the significative presence of highly pathogenic strains. Aeromonas strains possess a heterogeneous armamentarium of pathogenicity factors that allows the microbe to affect a wide range of human intestinal epithelial cell processes that justify the ability to induce diarrhea through different mechanisms and cause diseases of variable severity, as observed for other gastrointestinal pathogens. However, it remains to be determined whether specific genotype(s) are associated with clinical pictures of different severity to implement the diagnostic and therapeutic approaches for this relevant enteric pathogen.

Pretreatment with propofol restores intestinal epithelial cells integrity disrupted by mast cell degranulation in vitro

Propofol has been shown to against intestinal reperfusion injury when treated either before or after ischemia, during which mast cell could be activated. The aim of this study was to evaluate the role of propofol in restoring the intestinal epithelial cells integrity disrupted by mast cell activation or the released tryptase after activation in vitro. We investigated the effect of: (1) tryptase on Caco-2 monolayers in the presence of PAR-2 inhibitor or propofol, (2) mast cell degranulation in a Caco-2/LAD-2 co-culture model in the presence of propofol, and (3) propofol on mast cell degranulation. Epithelial integrity was detected using transepithelial resistance (TER) and permeability to fluorescein isothiocyanate (FITC)-dextran (the apparent permeability coefficient, Papp). The expression of junctional proteins zonula occludens-1 (ZO-1/TJP1) and occludin were determined using western blot analysis and immunofluorescence microscopy. The intracellular levels of reactive oxidative species (ROS) and Ca2+ were measured using flow cytometry. Tryptase directly enhanced intestinal barrier permeability as demonstrated by significant reductions in TER, ZO-1, and occludin protein expression and concomitant increases in Papp. The intestinal barrier integrity was restored by PAR-2 inhibitor but not by propofol. Meanwhile, mast cell degranulation resulted in epithelial integrity disruption in the Caco-2/LAD-2 co-culture model, which was dramatically attenuated by propofol. Mast cell degranulation caused significant increases in intracellular ROS and Ca(2+) levels, which were blocked by propofol and NAC. Propofol pretreatment can inhibit mast cell activation via ROS/Ca(2+) and restore the intestinal barrier integrity induced by mast cell activation, instead of by tryptase.

Barrier Perturbation in Porcine Peyer's Patches by Tumor Necrosis Factor is Associated With a Dysregulation of Claudins.

The proinflammatory cytokine tumor necrosis factor (TNF) has been described as one of the main mediators of intestinal inflammatory diseases, affecting the composition of tight junction (TJ) proteins and leading to a disruption of the epithelial barrier. An intact intestinal barrier is mandatory, because the follicle-associated epithelium of Peyer’s patches represents the first defense line of the intestinal immune system and ensures a controlled uptake of antigens from the gut lumen. In the current study, we have analyzed the detailed effects of TNF on the follicle-associated epithelium of porcine Peyer’s patches by applying the Ussing chamber technique. Epithelial tissue specimens of Peyer’s patches and the surrounding villus epithelium were mounted into conventional Ussing chambers and incubated with TNF for 10 h. The transepithelial resistance, representing epithelial barrier function of the tissue, was recorded. A reduction of transepithelial resistance was detected after 8 h in Peyer’s patch tissue specimens, whereas the villus epithelium was not significantly affected by TNF. Subsequent molecular analysis of TJ protein expression revealed a marked decrease of claudin-1 and -4, and an increase of claudin-2. In neighboring villus epithelium, no significant changes in the expression of TJ proteins could be shown. A strong increase of TNF receptor-2 (TNFR-2) could also be detected in Peyer’s patches, in agreement with the major role of this receptor in Peyer’s patches. Our findings were in accordance with changes detected by confocal laser scanning immunofluorescence microscopy. The regulation of TNF effects via myosin light chain kinase (MLCK) was analyzed in blocking experiments. Our detailed analysis is the first to show that TNF affects the barrier function of the follicle-associated epithelium of porcine Peyer’s patches but has no effects on the villus epithelium. These findings reveal not only the basic differences of epithelial barrier function between the two structures, but also the significance of Peyer’s patches as a primary mucosal immune defense.

Intestinal health in Atlantic salmon post-smolt (Salmo salar) when fed low- and high HUFA diets

It is well established that farmed Atlantic salmon (Salmo salar) need n3-highly unsaturated fatty acids (HUFA) in their diet to thrive and grow. However, the biological functions to the individual HUFAs may differ, implying that future supplementation could require fixed ratios for maximum benefit. The intestinal barrier is essential to fish health, and any disruption of the barrier can have detrimental effects. The current experiment was designed to examine the response of the intestinal hindgut when fed a low HUFA diet with 8.5 g/kg EPA + DHA (4.5% total fatty acid) or two high HUFA diets, with either high DHA (28.7 g/kg and 5.9% total fatty acid) or high EPA (25 g/kg and 14.2% total fatty acid). The diets were fed to Atlantic salmon post-smolt over 10 weeks and thereafter exposed to 3 weeks of chronic stress. After 10 weeks of feeding there were no differences in intestinal permeability and integrity, but intestinal morphology indicated increased intestinal health in the high EPA group. Gene expression also suggest that fish fed the high EPA diet had more regulation of pathways related to protein turnover compared to the high DHA fed fish. There was also indication of lower energy utilization in the low HUFA fed fish than high HUFA. Subjecting fish to 3 weeks of chronic stress led to a reduction in transepithelial resistance, increased ion flux and active l-lysine transport across the intestinal barrier in addition to a decrease in mucosal fold, enterocyte height and supranuclear vacuole density and an increase in thickness of the intestinal muscularis. After stress, the low HUFA group showed signs of inflammation with increased infiltration of MHCII positive cells. Gene expression also showed that low HUFA fed fish had a lower response to chronic stress compared to the high HUFA groups. Comparing fish fed either high DHA or EPA exposed to chronic stress showed few physical effects, but a lower density of supranuclear vacuoles and upregulation in immune-related gene expression indicate inflammation in the high DHA group.

Gastroesophageal Junction Fat Pad Tissue from Obese Patients Impairs Esophageal Epithelial Barrier Function and Disrupts Cell‐to‐Cell Connections

Introduction Visceral obesity is associated with GERD and, traditionally, it has been assumed that visceral fat predisposes to GERD primarily by increasing intra-abdominal pressure. However, transmission electron microscopy (TEM) of esophageal biopsies from obese individuals without GERD exhibit dilated intercellular spaces (DIS) and, in earlier studies, we found that substances secreted by mature adipocytes in visceral fat from obese patients impair esophageal epithelial barrier function, induce DIS and reduce transepithelial resistance (TER). There is a fat pad at the gastroesophageal junction (GEJ) that is in intimate contact with the distal esophagus. We now explore whether whole GEJ fat tissue of obese individuals secretes substances that impair esophageal barrier function. Methods We obtained GEJ fat from 3 obese (BMI 46, 60, 74) and 3 non-obese (BMI 24, 26, 27) patients having foregut surgery. Human esophageal squamous cells were seeded into transwells and grown to confluence for 3 days. On day 4, cells were raised to an air-liquid interface (ALI) to allow for differentiation, stratification, and esophageal barrier formation. ALI cultures were exposed to conditioned media (CM) from GEJ fat or to control medium beginning at day 4. TER was measured on days 4, 6, and 8 using the EVOM2 Epithelial Volt/Ohm Meter. On day 8, ALI cultures were stained with H&E and Ki-67 and imaged; barrier permeability was assessed by a biotinylation reagent applied to the ALI surface. TEM of ALIs and of esophageal biopsies from 2 non-obese and 2 obese patients without GERD were assessed. Results CM from all 3 obese patients significantly reduced TER on days 6 and 8; CM from 2 non-obese patients did not reduce TER whereas CM from 1 non-obese patient induced a slight reduction in TER at day 8 (Table 1). Marked DIS were found only in ALI cultures grown with CM from obese patients. In ALIs grown with CM from non-obese patients, the biotinylation reagent stopped at the superficial layers of esophageal cells. In contrast, the biotinylation reagent diffused through all cell layers in ALIs grown with obese patient CM. Increases in basaloid cells and in Ki-67 were found in ALIs grown with obese patient CM. TEMs of ALIs grown with non-obese patient CM demonstrated well-oriented cell-cell junctions similar to TEMs of non-obese patient esophageal biopsies. In contrast, ALIs grown with obese patient CM demonstrated cell-cell junctional structures that were disorganized, aggregated, and floating within the DIS, similar in appearance to TEMs of the obese patients’ esophageal biopsies. Conclusion Substances produced by GEJ fat of obese patients impair esophageal epithelial barrier integrity, cause basal cell hyperplasia and DIS, and disrupt intercellular connections. These findings in ALI cultures recapitulate abnormalities seen by TEM in esophageal biopsies of obese patients.

J or H mtDNA haplogroups in retinal pigment epithelial cells: Effects on cell physiology, cargo in extracellular vesicles, and differential uptake of such vesicles by naïve recipient cells

PurposeExtracellular vesicles (EVs) are predicted to represent the internal state of cells. In polarized RPE monolayers, EVs can mediate long-distance communication, requiring endocytosis via protein-protein interactions. EV uptake from oxidatively stressed donor cells triggers loss in transepithelial resistance (TER) in recipient monolayers mediated by HDAC6. Here, we examine EVs released from RPE cells with identical nuclear genes but different mitochondrial (mt)DNA haplogroups (H, J). J-cybrids produce less ATP, and the J-haplogroup is associated with a higher risk for age-related macular degeneration. MethodsCells were grown as mature monolayers to either collect EVs from apical surfaces or to serve as naïve recipient cells. Transfer assays, transferring EVs to a recipient monolayer were performed, monitoring TER and EV-uptake. The presence of known EV surface proteins was quantified by protein chemistry. ResultsH- and J-cybrids were confirmed to exhibit different levels of TER and energy metabolism. EVs from J-cybrids reduced TER in recipient ARPE-19 cells, whereas EVs from H-cybrids were ineffective. TER reduction was mediated by HDAC6 activity, and EV uptake required interaction between integrin and its ligands and surface proteoglycans. Protein quantifications confirmed elevated levels of fibronectin and annexin A2 on J-cybrid EVs. ConclusionsWe speculate that RPE EVs have a finite set of ligands (membrane proteoglycans and integrins and/or annexin A2) that are elevated in EVs from stressed cells; and that if EVs released by the RPE could be captured from serum, that they might provide a disease biomarker of RPE-dependent diseases.

Combined Bacteriophage and Antibiotic Treatment Prevents Pseudomonas aeruginosa Infection of Wild Type and cftr- Epithelial Cells.

With the increase of infections due to multidrug resistant bacterial pathogens and the shortage of antimicrobial molecules with novel targets, interest in bacteriophages as a therapeutic option has regained much attraction. Before the launch of future clinical trials, in vitro studies are required to better evaluate the efficacies and potential pitfalls of such therapies. Here we studied in an ex vivo human airway epithelial cell line model the efficacy of phage and ciprofloxacin alone and in combination to treat infection by Pseudomonas aeruginosa. The Calu-3 cell line and the isogenic CFTR knock down cell line (cftr-) infected apically with P. aeruginosa strain PAO1 showed a progressive reduction in transepithelial resistance during 24 h. Administration at 6 h p.i. of single phage, phage cocktails or ciprofloxacin alone prevented epithelial layer destruction at 24 h p.i. Bacterial regrowth, due to phage resistant mutants harboring mutations in LPS synthesis genes, occurred thereafter both in vitro and ex vivo. However, co-administration of two phages combined with ciprofloxacin efficiently prevented PAO1 regrowth and maintained epithelial cell integrity at 72 p.i. The phage/ciprofloxacin treatment did not induce an inflammatory response in the tested cell lines as determined by nanoString® gene expression analysis. We conclude that combination of phage and ciprofloxacin efficiently protects wild type and cftr- epithelial cells from infection by P. aeruginosa and emergence of phage resistant mutants without inducing an inflammatory response. Hence, phage-antibiotic combination should be a safe and promising anti-Pseudomonas therapy for future clinical trials potentially including cystic fibrosis patients.

Transient receptor potential V4 channel stimulation induces reversible epithelial cell permeability in MDCK cell monolayers.

The transient receptor potential V4 channel (TRPV4) is responsive to a variety of physical and chemical stimuli, including a synthetic agonist GSK1016790A (GSK). Here, we show that TRPV4 is functionally expressed in, and that GSK induces the reversible opening of tight junctions (TJs) in epithelial Madin-Darby canine kidney II monolayers. Stimulation of TRPV4 by GSK induces an increase in fluorescein isothiocyanate-conjugated dextran (4kDa) permeability and a reduction in transepithelial resistance, and these responses are blocked by pretreatment with the specific TRPV4 antagonist. Small conductance, but not large conductance Ca2+ -activated K+ channels, TRPA1 channel, and cofilin activation are involved in TRPV4-mediated reversible opening of TJs. These results suggest that a novel mechanism underlies TRPV4-mediated regulation of the tightness of epithelial barriers.

Basolateral pressure challenges mammary epithelial cell monolayer integrity, in vitro.

Mammary gland epithelium is physiologically exposed to variations of hydrostatic pressure due to accumulation of milk and removal by suckling and mechanical milking. Integrity of the mammary gland epithelium primarily relies on the tight junction. To analyze pressure-induced effects on the tight junction, we established a modified Ussing chamber and tested the hypothesis if hydrostatic pressure on the basal side of the epithelium is able to affect barrier properties in a mammary epithelial cell model, in vitro. Therefore, a conventional Ussing chamber was modified by an additional tube system to apply hydrostatic pressure. Monolayers of the mammary epithelial cell line HC11 were mounted in the modified Ussing chambers and incubated with increasing basal hydrostatic pressure. Transepithelial resistance and short circuit current were recorded and compared to controls. Hydrostatic pressure was stably applied and incubation steps of 30min were technically feasible, leading to a decrease of transepithelial resistance and an increase of short circuit current in all monolayers. In a series of experiments simulating the physiological exposure time by short intervals of 5min, these electrophysiological findings were also observed, and monolayer integrity was not significantly perturbed as analyzed by fluorescence immunohistochemistry selectively staining tight junction proteins. Moreover, electrophysiology demonstrated reversibility of effects. In conclusion, the modified Ussing chamber is an adequate method to analyze the effects of hydrostatic pressure on epithelial cell monolayers, in vitro. Both, the reduction of transepithelial resistance and the increase of short circuit current may be interpreted as protective reactions.

Chitosan hydrochloride has no detrimental effect on bladder urothelial cancer cells

Bladder cancer is among the most common and aggressive human malignant carcinomas, thus targeting and removal of bladder cancer cells is still a challenge. Although it is well known that chitosan hydrochloride (CH-HCl) causes desquamation of normal urothelial cells, its effect on cancer urothelial cells has not been recognized yet.In this in vitro study, we analyzed the cytotoxicity of 0.05% CH-HCl on three urothelial models: two cancer urothelial models, i.e. invasive and papillary urothelial neoplasms, and a normal urothelial model. The cytotoxicity of CH-HCl was evaluated with viability tests, transepithelial resistance (TER) measurements, and electron microscopy.TER measurements showed that 15-minute treatment with CH-HCl caused no reduction in TER of the cancer models, whereas the TER of the normal urothelial model significantly decreased. Furthermore, after CH-HCl treatment, the viability of cancer cells was reduced by only 5%, whereas the viability of normal cells was reduced by 30%. Ultrastructural analysis revealed necrotic cell death in all cases. We have demonstrated that although CH-HCl increases the mortality of cancer urothelial cells, it increases the mortality of normal urothelial cells even more so. However, shorter 2-minute CH-HCl treatment only temporarily increases the permeability of normal urothelial model, i.e. disrupts tight junctions and reduces TER without comprising cell viability, and enables the complete recovery of the permeability barrier after 24h.Overall, our results suggest that CH-HCl cannot be used as a self-sufficient anticancer agent for urothelial bladder cancer treatment; nevertheless a possibility of its use as an enhancer of cytostatic treatment is discussed.

A comparison of three Peyer's patch "M-like" cell culture models: particle uptake, bacterial interaction, and epithelial histology.

Intestinal Peyer's patch (PP) microfold (M) cells transport microbes and particulates across the follicle-associated epithelium (FAE) as part of the mucosal immune surveillance system. In vitro human M-like cell co-culture models are used as screens to investigate uptake of antigens-in-nanoparticles, but the models are labour-intensive and there is inter-laboratory variability. We compared the three most established filter-grown Caco-2/Raji B cell co-culture systems. These were Model A (Kernéis et al., 1997), Model B (Gullberg et al., 2000), and Model C (Des Rieux et al. 2007). The criteria used were transepithelial resistance (TEER), the apparent permeability coefficient (Papp) of [14C]-mannitol, M cell-like histology, as well as latex particle and Salmonella typhimurium translocation. Each co-culture model displayed substantial increases in particle translocation. Truncated microvilli compared to mono-cultures was their most consistent feature. The inverted model developed by des Rieux et al. (2007) displayed reductions in TEER and an increased (Papp), accompanied by the largest increase in particle translocation compared to the other two models. The normally-oriented model developed by Gullberg et al. (2000) was the only one to consistently display an increased translocation of Salmonella typhimurium. By applying a double Matrigel™ coating on filters, altering the medium feeding regime for Raji B cells, and restricting the passage number of B cells, improvements to the Gullberg model B were achieved, as reflected by increased particle translocation and improved histology. In conclusion, this is the first time all three designs have been compared in one study and each displays phenotypic features of M-like cells. While Model C was the most robust co-culture, the Model B protocol could be improved by optimizing several variables and is less complicated to establish than the two inverted models.

BLT2 expression improves skin integrity and protects from alterations caused by hyperglycemia in type 2 diabetes.

ABSTRACTType 2 diabetes (T2D) can go undiagnosed for years, leading to a stage where chronic high blood sugar produces complications such as delayed wound healing. Reports have shown that BLT2 activation improves keratinocyte migration and wound healing, as well as protecting the epidermal barrier through the promotion of actin polymerization.The goal of this study was to elucidate the role of BLT2 expression in skin epithelial integrity in T2D.For this purpose, we used both wild type (WT) and BLT2 knockout mice in a model, in which a T2D-like phenotype was induced by keeping the animals on a high fat (HF) diet over 5 weeks. In a parallel in vitro approach, we cultured BLT2-transfected HaCaT cells at both low and high glucose concentrations for 48 h. Structure, transepithelial resistance (TEER), IL-1ß, IL-8 or CXCL2, MMP9, Filaggrin, Loricrin and Keratin 10 (K10) were evaluated ex vivo and in vitro. Additionally, wound healing (WH) was studied in vitro.The skin from T2D and BLT2 knockout mice showed a reduction in TEER and the expression of IL-1ß, and in increase in CXCL2, MMP9, Filaggrin, Loricrin and K10 expression. The structure suggested an atrophic epidermis; however, the skin was dramatically affected in the BLT2 knockout mice kept on a HF diet.HaCaT-BLT2 cells presented as an organized monolayer and showed higher TEER and wound healing compared with vector only-transfected HaCaT-Mock cells. Likewise, alterations in the expression of skin inflammatory, matrix degradation and differentiation markers under low and high glucose conditions were less severe than in HaCaT-Mock cells.Our results suggest that BLT2 improves epithelial integrity and function by regulating differentiation markers, cytokines and MMP9. Furthermore, BLT2 attenuates the damaging effects of high glucose levels, thereby accelerating wound healing.

The Modulatory Effect of Common Dietary Fatty Acids on IPEC‐J2 Transport of Lipopolysaccharide and Monolayer Barrier Integrity in vitro

Recent evidence has demonstrated the ability of dietary fatty acid composition to influence postprandial endotoxemia in humans and swine. However, the mechanism(s) by which fatty acids affect transport of lipopolysaccharide (LPS) across the intestinal epithelium has not yet been fully elucidated. To study this, we utilized non‐transformed porcine jejunum‐derived IPEC‐J2 cells. This cell line is well‐recognized to form confluent polarized monolayers with apical and basolateral regions, allowing for an in vitro model of the intestinal epithelium. The present study investigated the effect dietary fatty acids and inflammation had on intestinal permeability of LPS and barrier integrity in IPEC‐J2 monolayers. We hypothesized that treatment of IPEC‐J2 monolayer with saturated or n‐6 polyunsaturated fatty acids (PUFA) would enhance fluorescein‐isothiocyanate (FITC)‐LPS apical‐to‐basolateral permeability and reduce transepithelial resistance (TER); whereas n‐3 PUFA would attenuate this response. Secondly, we hypothesized that lauric acid would amplify the reduction in monolayer integrity associated with pre‐treatment with LPS. IPEC‐J2 cell monolayers were grown to confluency (>1kΩ) on 0.4 μm Transwell® inserts. In experiment 1, these monolayers were treated with either 50 or 100 μM of docosahexaenoic acid, lauric acid, arachidonic acid or vehicle for 48 hours before TER and FITC‐LPS determination of barrier integrity. In experiment 2, monolayers were treated with either 5 μg/mL LPS, LPS plus 100 μM lauric acid or saline vehicle for 24 hours and then TER and FITC‐LPS determination of barrier integrity assessed. In experiment 1, TER and FITC‐LPS permeability was not altered by fatty acid type or concentration. For experiment 2, compared to the saline control, LPS caused a significant time‐dependent reduction in TER (p<0.001); however, the co‐inclusion of lauric acid fatty acids did not have any additive effect on TER (p>0.05). Although TER was reduced, surprisingly we reported no treatment differences in apical to basolateral FITC‐LPS permeability. In conclusion, fatty acid treatment did not modulate barrier inflammation and permeability in in vitro IPEC‐J2 monolayers. However, live Gram‐negative bacteria or their bacterial cellular membrane fragments may be a better model to study the effects of fatty acids on LPS permeability.Support or Funding InformationResearch supported by the Agriculture and Food Research Initiative Grant No. 2014‐67017‐21778 from the USDA NIFA.

Nod-Like Receptor X-1 Is Required for Rhinovirus-Induced Barrier Dysfunction in Airway Epithelial Cells

Barrier dysfunction of airway epithelium may increase the risk for acquiring secondary infections or allergen sensitization. Both rhinovirus (RV) and polyinosinic-polycytidilic acid [poly(I·C)], a double-stranded RNA (dsRNA) mimetic, cause airway epithelial barrier dysfunction, which is reactive oxygen species (ROS) dependent, implying that dsRNA generated during RV replication is sufficient for disrupting barrier function. We also demonstrated that RV or poly(I·C)-stimulated NADPH oxidase 1 (NOX-1) partially accounts for RV-induced ROS generation. In this study, we identified a dsRNA receptor(s) contributing to RV-induced maximal ROS generation and thus barrier disruption. We demonstrate that genetic silencing of the newly discovered dsRNA receptor Nod-like receptor X-1 (NLRX-1), but not other previously described dsRNA receptors, abrogated RV-induced ROS generation and reduction of transepithelial resistance (R(T)) in polarized airway epithelial cells. In addition, both RV and poly(I·C) stimulated mitochondrial ROS, the generation of which was dependent on NLRX-1. Treatment with Mito-Tempo, an antioxidant targeted to mitochondria, abolished RV-induced mitochondrial ROS generation, reduction in R(T), and bacterial transmigration. Furthermore, RV infection increased NLRX-1 localization to the mitochondria. Additionally, NLRX-1 interacts with RV RNA and poly(I·C) in polarized airway epithelial cells. Finally, we show that NLRX-1 is also required for RV-stimulated NOX-1 expression. These findings suggest a novel mechanism by which RV stimulates generation of ROS, which is required for disruption of airway epithelial barrier function. Rhinovirus (RV), a virus responsible for a majority of common colds, disrupts the barrier function of the airway epithelium by increasing reactive oxygen species (ROS). Poly(I·C), a double-stranded RNA (dsRNA) mimetic, also causes ROS-dependent barrier disruption, implying that the dsRNA intermediate generated during RV replication is sufficient for this process. Here, we demonstrate that both RV RNA and poly(I·C) interact with NLRX-1 (a newly discovered dsRNA receptor) and stimulate mitochondrial ROS. We show for the first time that NLRX-1 is primarily expressed in the cytoplasm and at the apical surface rather than in the mitochondria and that NLRX-1 translocates to mitochondria following RV infection. Together, our results suggest a novel mechanism for RV-induced barrier disruption involving NLRX-1 and mitochondrial ROS. Although ROS is necessary for optimal viral clearance, if not neutralized efficiently, it may increase susceptibility to secondary infections and alter innate immune responses to subsequently inhaled pathogens, allergens, and other environmental factors.

Providencia alcalifaciens causes barrier dysfunction and apoptosis in tissue cell culture: potent role of lipopolysaccharides on diarrheagenicity

Providencia alcalifaciens is a member of the Enterobacteriaceae family that occasionally causes diarrheagenic illness in humans via the intake of contaminated foods. Despite the epidemiological importance of P. alcalifaciens, little is known about its pathobiology. Here we report that P. alcalifaciens causes barrier dysfunction in Caco-2 cell monolayers and induces apoptosis in calf pulmonary artery endothelial cells. P. alcalifaciens infection caused a 30% reduction in transepithelial resistance in Caco-2 cell monolayers, which was greater than that for cells infected with Shigella flexneri or non-pathogenic Escherichia coli. As with viable bacteria, bacterial lysates treated with heat, benzonase or proteinase, but not with polymixin B, were also involved in the cellular response. TLR4 antibody neutralisation significantly restored the P. alcalifaciens-induced transepithelial resistance reduction in Caco-2 cells, suggesting that lipopolysaccharides (LPSs) might play a central role in this cellular response. Western blotting further indicated that P. alcalifaciens LPSs reduced occludin levels, whereas LPSs from Shigella or E. coli did not. Although the viability of Caco-2 cells was not altered significantly, the calf pulmonary artery endothelial cell line was highly sensitive to P. alcalifaciens infection. This sensitivity was indeed dependent on LPS, which induced rapid apoptosis. Together, these data show that P. alcalifaciens LPSs participate in epithelial barrier dysfunction and endothelial apoptosis. The findings give insight into the LPS-dependent cell signal events affecting diarrheagenicity during infection with P. alcalifaciens.

Basolateral BMP Signaling in Polarized Epithelial Cells

Bone morphogenetic proteins (BMPs) regulate various biological processes, mostly mediated by cells of mesenchymal origin. However, the roles of BMPs in epithelial cells are poorly understood. Here, we demonstrate that, in polarized epithelial cells, BMP signals are transmitted from BMP receptor complexes exclusively localized at the basolateral surface of the cell membrane. In addition, basolateral stimulation with BMP increased expression of components of tight junctions and enhanced the transepithelial resistance (TER), counteracting reduction of TER by treatment with TGF-β or an anti-tumor drug. We conclude that BMPs maintain epithelial polarity via intracellular signaling from basolaterally localized BMP receptors.

Oxidative Stress Sensitizes Retinal Pigmented Epithelial (RPE) Cells to Complement-mediated Injury in a Natural Antibody-, Lectin Pathway-, and Phospholipid Epitope-dependent Manner

Uncontrolled activation of the alternative complement pathway (AP) is thought to be associated with age-related macular degeneration. Previously, we have shown that in retinal pigmented epithelial (RPE) monolayers, oxidative stress reduced complement inhibition on the cell surface, resulting in sublytic complement activation and loss of transepithelial resistance (TER), but the potential ligand and pathway involved are unknown. ARPE-19 cells were grown as monolayers on transwell plates, and sublytic complement activation was induced with H2O2 and normal human serum. TER deteriorated rapidly in H2O2-exposed monolayers upon adding normal human serum. Although the effect required AP activation, AP was not sufficient, because elimination of MASP, but not C1q, prevented TER reduction. Reconstitution experiments to unravel essential components of the lectin pathway (LP) showed that both ficolin and mannan-binding lectin can activate the LP through natural IgM antibodies (IgM-C2) that recognize phospholipid cell surface modifications on oxidatively stressed RPE cells. The same epitopes were found on human primary embryonic RPE monolayers. Likewise, mouse laser-induced choroidal neovascularization, an injury that involves LP activation, could be increased in antibody-deficient rag1(-/-) mice using the phospholipid-specific IgM-C2. In summary, using a combination of depletion and reconstitution strategies, we have shown that the LP is required to initiate the complement cascade following natural antibody recognition of neoepitopes, which is then further amplified by the AP. LP activation is triggered by IgM bound to phospholipids. Taken together, we have defined novel mechanisms of complement activation in oxidatively stressed RPE, linking molecular events involved in age-related macular degeneration, including the presence of natural antibodies and neoepitopes.

Lipopolysaccharide-induced phosphorylation of c-Met tyrosine residue 1003 regulates c-Met intracellular trafficking and lung epithelial barrier function

c-Met, the receptor tyrosine kinase whose natural ligand is hepatocyte growth factor, is known to have a key role in cell motility. We have previously shown that lysophosphatidic acid (LPA) induced a decrease in c-Met activation via serine phosphorylation of c-Met at cell-cell contacts. Here, we demonstrate that lipopolysaccharide (LPS) treatment of human bronchial epithelial cells induced internalization of c-Met via phosphorylation at its tyrosine residue 1003. In addition, it induced epithelial barrier dysfunction as evidenced by a decrease in transepithelial resistance (TER) in a time-dependent manner. Pretreatment with a c-Met inhibitor (PHA-665752) or inhibition of protein kinase C (PKC)-α attenuated the LPS-mediated phosphorylation of c-Met and its internalization. LPS-induced c-Met tyrosine 1003 phosphorylation, activation of PKCα, and c-Met internalization were, however, reversed by pretreatment of cells with LPA, which increased c-Met accumulation at cell-cell contacts. Inhibition of LPS-mediated c-Met tyrosine (Y1003) phosphorylation and internalization by prior treatment with PHA-665752, inhibition of PKCα, or overexpression of c-MetY1003A mutant attenuated LPS-induced reduction of TER. Furthermore, we found that c-Met accumulation at cell-cell contacts contributed to LPA-enhanced epithelial barrier integrity, since downregulation of c-Met by specific small-interfering RNA attenuated LPA-increased TER. The data reveal a novel biological function of c-Met in the regulation of lung epithelial barrier integrity.

A role for tricellulin in the regulation of gill epithelium permeability

The apical-most region of cell-to-cell contact in a vertebrate epithelium is the tight junction (TJ) complex. It is composed of bicellular TJs (bTJs) that bridge two adjacent epithelial cells and tricellular TJs (tTJs) that are points of contact between three adjoining epithelial cells. Tricellulin (TRIC) is a transmembrane TJ protein of vertebrates that is found in the tTJ complex. Full-length cDNA encoding rainbow trout TRIC was cloned and sequenced. In silico analysis of rainbow trout TRIC revealed a tetraspannin protein with several putative posttranslational modification sites. TRIC mRNA was broadly expressed in rainbow trout tissues and exhibited moderately greater abundance in the gill. In a primary cultured gill epithelium, TRIC localized to tTJs and TRIC protein abundance increased in association with corticosteroid-induced reductions in paracellular permeability. Sodium caprate was used to compromise cultured gill epithelium integrity by disrupting the tTJ complex. Sodium caprate treatment caused a reversible reduction in transepithelial resistance, caused an increase in paracellular permeability (as measured by [³H]PEG-4000 flux), and displaced TRIC from tTJs while leaving bTJs intact. Data from this study support the view that tTJs and the TJ protein TRIC 1) play a role in maintaining gill epithelium integrity and 2) contribute to the regulation of gill epithelium permeability.

Chymase-mediated intestinal epithelial permeability is regulated by a protease-activating receptor/matrix metalloproteinase-2-dependent mechanism.

Mast cells regulate intestinal barrier function during disease and homeostasis. Secretion of the mast cell-specific serine protease chymase regulates homeostasis. In the present study, we employ in vitro model systems to delineate the molecular pathways involved in chymase-mediated intestinal epithelial barrier dysfunction. Chymase stimulation of intestinal epithelial (Caco-2 BBe) cell monolayers induced a significant reduction in transepithelial resistance, indicating decreased intestinal epithelial barrier function. The chymase-induced intestinal epithelial barrier dysfunction was characterized by chymase-induced protease-activated receptor (PAR)-2 activation and matrix metalloproteinase (MMP)-2 expression and activation. Consistent with this observation, in vitro analysis revealed chymase-induced PAR-2 activation and increased MAPK activity and MMP-2 expression. Pharmacological and small interfering RNA-mediated antagonism of PAR-2 and MMP-2 significantly attenuated chymase-stimulated barrier dysfunction. Additionally, the chymase/MMP-2-mediated intestinal epithelial dysfunction was associated with a significant reduction in the tight junction protein claudin-5, which was partially restored by MMP-2 inhibition. Finally, incubation of Caco-2 BBe cells with chymase-sufficient, but not chymase-deficient, bone marrow-derived mast cells decreased barrier function, which was attenuated by the chymase inhibitor chymostatin. Collectively, these results suggest that mast cell/chymase-mediated intestinal epithelial barrier function is mediated by PAR-2/MMP-2-dependent pathways.