Quercetin Improves Barrier Properties in Porcine Small Intestine but Not in Peyer's Patches.

Peyer's patches (PPs) are part of the gut-associated lymphatic tissue (GALT) and represent the first line of the intestinal immunological defense. They consist of follicles with lymphocytes and an overlying subepithelial dome with dendritic cells and macrophages, and they are covered by the follicle-associated epithelium (FAE). A sealed paracellular pathway in the FAE is crucial for the controlled uptake of luminal antigens. Quercetin is the most abundant plant flavonoid and has a barrier-strengthening effect on tight junctions (TJs), a protein complex that regulates the paracellular pathway. In this study, we aimed to analyze the effect of quercetin on porcine PPs and the surrounding villus epithelium (VE). We incubated both tissue types for 4 h in Ussing chambers, recorded the transepithelial electrical resistance (TEER), and measured the unidirectional tracer flux of [3H]-mannitol. Subsequently, we analyzed the expression, protein amount, and localization of three TJ proteins, claudin 1, claudin 2, and claudin 4. In the PPs, we could not detect an effect of quercetin after 4 h, neither on TEER nor on the [3H]-mannitol flux. In the VE, quercetin led to a higher TEER value, while the [3H]-mannitol flux was unchanged. The pore-forming claudin 2 was decreased while the barrier-forming claudin 4 was increased and the expression was upregulated. Claudin 1 was unchanged and all claudins could be located in the paracellular membrane by immunofluorescence microscopy. Our study shows the barrier-strengthening effect of quercetin in porcine VE by claudin 4 upregulation and a claudin 2 decrease. Moreover, it underlines the different barrier properties of PPs compared to the VE.

Cellular Distribution Pattern of tjp1 (ZO-1) in Xenopus laevis Oocytes Heterologously Expressing Claudins.

Epithelial barriers constitute a fundamental requirement in every organism, as they allow the separation of different environments and set boundaries against noxious and other adverse effectors. In many inflammatory and degenerative diseases, epithelial barrier function is impaired because of a disturbance of the paracellular seal. Recently, the Xenopus laevis oocyte has been established as a heterologous expression model for the analysis of transmembrane tight junction protein interactions and is currently considered to be a suitable screening model for barrier effectors. A prerequisite for this application is a physiological anchoring of claudins to the cytoskeleton via the major scaffolding protein tjp1 (tight junction protein 1, ZO-1). We have analyzed the oocyte model with regard to the interaction of heterologously expressed claudins and tjp1. Our experiments have revealed endogenous tjp1 expression in protein and mRNA analyses of unfertilized Xenopus laevis oocytes expressing human claudin 1 (CLDN1) to claudin 5 (CLDN5). The amphibian cell model can therefore be used for the analysis of claudin interactions.

Prednisolone Targets Claudins in Mouse Brain Blood Vessels.

Endothelial cells in brain capillaries are crucial for the function of the blood-brain barrier (BBB), and members of the tight junction protein family of claudins are regarded to be primarily responsible for barrier properties. Thus, the analysis of bioactive substances that can affect the BBB's permeability is of great importance and may be useful for the development of new therapeutic strategies for brain pathologies. In our study, we tested the hypothesis that the application of the glucocorticoid prednisolone affects the murine blood-brain barrier in vivo. Isolated brain tissue of control and prednisolone-injected mice was examined by employing immunoblotting and confocal laser scanning immunofluorescence microscopy, and the physiological and behavioral effects were analyzed. The control tissue samples revealed the expression of barrier-forming tight junction proteins claudin-1, -3, and -5 and of the paracellular cation and water-channel-forming protein claudin-2. Prednisolone administration for 7 days at doses of 70 mg/kg caused physiological and behavioral effects and downregulated claudin-1 and -3 and the channel-forming claudin-2 without altering their localization in cerebral blood vessels. Changes in the expression of these claudins might have effects on the ionic and acid-base balance in brain tissue, suggesting the relevance of our findings for therapeutic options in disorders such as cerebral edema and psychiatric failure.

Selective Role of TNFα and IL10 in Regulation of Barrier Properties of the Colon in DMH-Induced Tumor and Healthy Rats.

Recently it has been reported that the tumor adjacent colon tissues of 1,2-dymethylhydrazine induced (DMH)-rats revealed a high paracellular permeability. We hypothesized that the changes might be induced by cytokines. Colorectal cancer is accompanied by an increase in tumor necrosis factor alpha (TNFα) and interleukin 10 (IL10) that exert opposite regulatory effects on barrier properties of the colon, which is characterized by morphological and functional segmental heterogeneity. The aim of this study was to analyze the level of TNFα and IL10 in the colon segments of DMH-rats and to investigate their effects on barrier properties of the proximal and distal parts of the colon in healthy rats. Enzyme immunoassay analysis showed decreased TNFα in tumors in the distal part of the colon and increased IL10 in proximal tumors and in non-tumor tissues. Four-hour intraluminal exposure of the colon of healthy rats with cytokines showed reduced colon barrier function dependent on the cytokine: TNFα decreased it mainly in the distal part of the colon, whereas IL10 decreased it only in the proximal part. Western blot analysis revealed a more pronounced influence of IL10 on tight junction (TJ) proteins expression by down-regulation of the TJ proteins claudin-1, -2 and -4, and up-regulation of occludin only in the proximal part of the colon. These data may indicate a selective role of the cytokines in regulation of the barrier properties of the colon and a prominent role of IL10 in carcinogenesis in its proximal part.

Effects of 1,2-Dimethylhydrazine on Barrier Properties of Rat Large Intestine and IPEC-J2 Cells.

Colon cancer is accompanied by a decrease of epithelial barrier properties, which are determined by tight junction (TJ) proteins between adjacent epithelial cells. The aim of the current study was to analyze the expression of TJ proteins in a rat model of 1,2-dimethylhydrazine (DMH)-induced colorectal cancer, as well as the barrier properties and TJ protein expression of IPEC-J2 cell monolayers after incubation with DMH. Transepithelial electrical resistance and paracellular permeability for sodium fluorescein of IPEC-J2 were examined by an epithelial volt/ohm meter and spectrophotometry. The expression and localization of TJ proteins were analyzed by immunoblotting and immunohistochemistry. In the colonic tumors of rats with DMH-induced carcinogenesis, the expression of claudin-3 and -4 was significantly increased compared to controls. The transepithelial electrical resistance of IPEC-J2 cells increased, while paracellular permeability for sodium fluorescein decreased, accompanied by an increased expression of claudin-4. The increase of claudin-4 in rat colon after chronic DMH exposure was consistent with the acute effect of DMH on IPEC-J2 cells, which may indicate an essential role of this protein in colorectal cancer development.

Tumor Necrosis Factor Alpha Effects on the Porcine Intestinal Epithelial Barrier Include Enhanced Expression of TNF Receptor 1.

Tumor necrosis factor alpha (TNFα) has been shown to impair the intestinal barrier, inducing and maintaining inflammatory states of the intestine. The aim of the current study was to analyze functional, molecular and regulatory effects of TNFα in a newly established non-transformed jejunal enterocyte model, namely IPEC-J2 monolayers. Incubation with 1000 U/mL TNFα induced a marked decrease in transepithelial electrical resistance (TEER), and an increase in permeability for the paracellular flux marker [3H]-D-mannitol compared to controls. Immunoblots revealed a significant decrease in tight junction (TJ) proteins occludin, claudin-1 and claudin-3. Moreover, a dose-dependent increase in the TNF receptor (TNFR)-1 was detected, explaining the exponential nature of pro-inflammatory effects, while TNFR-2 remained unchanged. Recovery experiments revealed reversible effects after the removal of the cytokine, excluding apoptosis as a reason for the observed changes. Furthermore, TNFα signaling could be inhibited by the specific myosin light chain kinase (MLCK) blocker ML-7. Results of confocal laser scanning immunofluorescence microscopy were in accordance with all quantitative changes. This study explains the self-enhancing effects of TNFα mediated by MLCK, leading to a differential regulation of TJ proteins resulting in barrier impairment in the intestinal epithelium.

Xenopus oocytes as a heterologous expression system for analysis of tight junction proteins.

Claudins (cldns) represent the largest family of transmembrane tight junction (TJ) proteins, determining organ-specific epithelial barrier properties. Because methods for the analysis of multiple cldn interaction are limited, we have established the heterologous Xenopus laevis oocyte expression system for TJ protein assembly and interaction analysis. Oocytes were injected with cRNA encoding human cldn-1, -2, or -3 or with a combination of these and were incubated in pairs for interaction analysis. Immunoblotting and immunohistochemistry were performed, and membrane contact areas were analyzed morphometrically and by freeze fracture electron microscopy. Cldns were specifically detected in membranes of expressing oocytes, and coincubation of oocytes resulted in adhesive contact areas that increased with incubation time. Adjacent membrane areas revealed specific cldn signals, including "kissing-point"-like structures representing homophilic trans-interactions of cldns. Contact areas of oocytes expressing a combination markedly exceeded those expressing single cldns, indicating effects on adhesion. Ultrastructural analysis revealed a self-assembly of TJ strands and a cldn-specific strand morphology.-Vitzthum, C., Stein, L., Brunner, N., Knittel, R., Fallier-Becker, P., Amasheh, S. Xenopus oocytes as a heterologous expression system for analysis of tight junction proteins.

Circulating Ouabain Modulates Expression of Claudins in Rat Intestine and Cerebral Blood Vessels.

The ability of exogenous low ouabain concentrations to affect claudin expression and therefore epithelial barrier properties was demonstrated previously in cultured cell studies. We hypothesized that chronic elevation of circulating ouabain in vivo can affect the expression of claudins and tight junction permeability in different tissues. We tested this hypothesis in rats intraperitoneally injected with ouabain (1 µg/kg) for 4 days. Rat jejunum, colon and brain frontal lobes, which are variable in the expressed claudins and tight junction permeability, were examined. Moreover, the porcine jejunum cell line IPEC-J2 was studied. In IPEC-J2-cells, ouabain (10 nM, 19 days of incubation) stimulated epithelial barrier formation, increased transepithelial resistance and the level of cSrc-kinase activation by phosphorylation, accompanied with an increased expression of claudin-1, -5 and down-regulation of claudin-12; the expression of claudin-3, -4, -8 and tricellulin was not changed. In the jejunum, chronic ouabain increased the expression of claudin-1, -3 and -5 without an effect on claudin-2 and -4 expression. In the colon, only down-regulation of claudin-3 was observed. Chronic ouabain protected the intestine transepithelial resistance against functional injury induced by lipopolysaccharide treatment or by modeled acute microgravity; this regulation was most pronounced in the jejunum. Claudin-1 was also up-regulated in cerebral blood vessels. This was associated with reduction of claudin-3 expression while the expression of claudin-5 and occludin was not affected. Altogether, our results confirm that circulating ouabain can functionally and tissue-specifically affect barrier properties of epithelial and endothelial tissues via Na,K-ATPase-mediated modulation of claudins expression.

Cholera toxin perturbs the paracellular barrier in the small intestinal epithelium of rats by affecting claudin-2 and tricellulin.

Cholera toxin is commonly known to induce chloride secretion of the intestine. In recent years, effects on epithelial barrier function have been reported, indicating synergistic co-regulation of transporters and tight junction proteins. Our current study focused on the analysis of cholera toxin effects on transepithelial resistance and on tight junction proteins, the latter known as structural correlates of barrier function. Ligated segments of the rat jejunum were injected with buffered solution containing cholera toxin (1 µg/ml) and incubated for 4 h. Subsequently, selfsame tissue specimens were mounted in Ussing chambers, and cholera toxin (1 µg/ml) was added on the apical side. Transepithelial resistance and permeability of sodium fluorescein (376 Da) were analyzed. Subsequently, tissues were removed, expression and localization of claudins were analyzed, and morphological studies were performed employing transmission electron microscopy and confocal laser scanning microscopy. Cholera toxin induced a marked decrease in transepithelial resistance in the rat jejunal epithelium and an increase in paracellular permeability for sodium fluorescein. Immunoblotting of tight junction proteins revealed an increase in claudin-2 signals, which was verified by confocal laser scanning immunofluorescence microscopy, and a decrease in tricellulin, whereas other tight junction proteins remained unchanged. Transmission electron microscopy showed a reduction in the number of microvilli after incubation with cholera toxin. Moreover, cholera toxin led to a widening of the intercellular space between enterocytes. In accordance with the commonly known prosecretory effect of cholera toxin, our study revealed a complementary effect on small intestinal barrier function and integrity, which might constitute a pathomechanism with high relevance for prevention and therapeutic approaches.

Modulation of gastrointestinal barrier and nutrient transport function in farm animals by natural plant bioactive compounds - A comprehensive review.

The use of antibiotics in diets has been restricted in several countries as a precautionary measure to avoid development of antibiotic resistance among pathogenic microorganisms. This regulation promoted the exploration of natural plant bioactive compounds (PBCs) as feed additives to improve productivity, welfare and health of livestock and poultry. Along with several beneficial attributes of PBCs, including antimicrobial, antioxidant and various pharmacological effects, they also improve barrier function and nutrient transport in the gastrointestinal (GI) tract. This comprehensive review discusses the effects of different PBCs on the integrity, nutrient transport and permeability of GI epithelia and their mechanism of actions. Dietary PBCs influence the maintenance and enhancement of GI integrity via a number of mechanisms including altered signaling pathways and expression of several tight junction proteins (claudins, occludin, and zonula occludens proteins), altered expression of various cytokines, chemokines, complement components and their transcription factors, goblet cell abundance and mucin gene expression, and the modulation of the cellular immune system. They also affect nutrient transporter gene expression and active absorption of nutrients, minerals and ammonia. One intriguing perspective is to select an effective dose at which a specific PBC could improve GI barrier function and nutrient absorption. The effective doses and clear-cut molecular mechanisms for PBCs are yet to be elucidated to understand discrepant observations among different studies and to improve the targeted biotechnological and pharmaceutical uses of PBCs in farm animals. The latter will also enable a more successful use of such PBCs in humans.

Symposium review: The importance of the ruminal epithelial barrier for a healthy and productive cow.

The stratified squamous ruminal epithelium is the main site for absorption of key nutrients (e.g., short-chain fatty acids; SCFA) and electrolytes (e.g., sodium and magnesium). The absorptive function has to be highly selective to prevent simultaneous entry of microbes and toxins from the rumen into the blood. As such, epithelial absorption is primarily transcellular, whereas the paracellular pathway appears rather tightly sealed. A network of tight junction (claudin-1, claudin-4, and occludin) and tight junction-associated proteins (e.g., zonula occludens) accomplishes the latter. When microbial fermentation activity is high such as with highly fermentable diets, rumen epithelial functions are often challenged by acidity, high osmolarity, toxins (e.g., endotoxin and histamine), and immune mediators (inflammatory mediators and cytokines) released during local and systemic inflammation. Epithelial damage by low pH in combination with high luminal SCFA concentrations is not immediately reversible and may initially aggravate upon return to physiological pH. In contrast, barrier opening upon hyperosmolarity is acutely transient. The initial insults set by luminal acidity and SCFA and the increasing concentrations of microbial-associated molecular patterns such as lipopolysaccharides are key factors that trigger inflammation not only in the rumen but also in the hindgut (cecum and colon), which reach out to the liver and other organs, causing systemic inflammation. Low feed intake during parturition, transportation, heat stress, or disease is the second most relevant challenge for the ruminal epithelial barrier. The barrier opening is usually only transient and quickly restored upon refeeding. Due to a rapid, dose-dependent, and prolonged decrease in absorption capacity for SCFA, however, any feed restriction increases the odds for postrestriction subacute ruminal acidosis. Inflammation due to acidosis can be alleviated by supplemental thiamine, yeasts, and plant bioactive (phytogenic) compounds. Butyrate is used in weaning calves to support ruminal barrier development; however, excess butyrate may promote hyperkeratosis, parakeratosis, and epithelial injury in the fully developed rumen of adult cows. Further research is needed to enhance the understanding of the various factors that counteract barrier impairment and help barrier restoration during acidogenic feeding, especially when concurring with unavoidable periods of feed restriction.

Caprate Modulates Intestinal Barrier Function in Porcine Peyer's Patch Follicle-Associated Epithelium.

BACKGROUND: Many food components influence intestinal epithelial barrier properties and might therefore also affect susceptibility to the development of food allergies. Such allergies are triggered by increased antibody production initiated in Peyer's patches (PP). Usually, the presentation of antigens in the lumen of the gut to the immune cells of the PP is strongly regulated by the follicle-associated epithelium (FAE) that covers the PP. As the food component caprate has been shown to impede barrier properties in villous epithelium, we hypothesized that caprate also affects the barrier function of the PP FAE, thereby possibly contributing a risk factor for the development of food allergies. METHODS: In this study, we have focused on the effects of caprate on the barrier function of PP, employing in vitro and ex vivo experimental setups to investigate functional and molecular barrier properties. Incubation with caprate induced an increase of transepithelial resistance, and a marked increase of permeability for the paracellular marker fluorescein in porcine PP to 180% of control values. These effects are in accordance with changes in the expression levels of the barrier-forming tight junction proteins tricellulin and claudin-5. CONCLUSIONS: This barrier-affecting mechanism could be involved in the initial steps of a food allergy, since it might trigger unregulated contact of the gut lumen with antigens.

Effects of glucagon-like peptides 1 and 2 and epidermal growth factor on the epithelial barrier of the rumen of adult sheep.

Epidermal growth factor (EGF) and glucagon-like peptides (GLP) modulate the tight junctions (TJ) of the intestinal epithelial barrier (EB) of monogastric animals. This work tried to elucidate whether GLP-1, GLP-2 and EGF can affect the EB of the rumen. Ovine ruminal epithelia were incubated in Ussing chambers for 7 hr with 25 or 250 nM of either GLP-1 or GLP-2 on the serosal side, with 2.5 nM of EGF on the serosal side or with 0.25 or 2.5 nM EGF on the mucosal side. No treatment affected tissue conductance. Short-circuit current (Isc ) was affected by time and treatment and their interactions. Only 250 nM of either GLP-1 or GLP-2 decreased Isc in certain periods compared with 25 nM GLP-1 or 0.25 nM mucosally applied EGF; however, not when compared to control epithelia. Fluorescein flux rates (Jfluor ) of ruminal epithelia were affected by treatment, time and time × treatment interaction. The time × treatment interaction was based on an increase in Jfluor between the first and last hour in epithelia incubated with 25 nM GLP-1 or GLP-2 and in epithelia incubated with EGF. After 7 hr incubation, claudin-7 mRNA expression was downregulated in all treatments. Claudin-1 mRNA was upregulated after incubation with 2.5 nM EGF on the serosal side, claudin-4 mRNA was downregulated by 2.5 nM EGF on the mucosal side, and occludin mRNA was increased after incubation with 250 nM GLP-2. The protein abundance of all tested TJ proteins was not influenced by treatment. We conclude that GLP-1, GLP-2, and EGF have no obvious acute effects on the EB of ruminal epithelia under simulated physiological conditions ex vivo. However, by decreasing the mRNA expression of claudin-7 and partly affecting other TJ proteins, they may modulate EB in the longer term or under certain conditions.

Formula Feeding Predisposes Neonatal Piglets to Clostridium difficile Gut Infection.

Spontaneous outbreaks of Clostridium difficile infection (CDI) occur in neonatal piglets, but the predisposing factors are largely not known. To study the conditions for C. difficile colonization and CDI development, 48 neonatal piglets were moved into isolators, fed bovine milk-based formula, and infected with C. difficile 078. Analyses included clinical scoring; measurement of the fecal C. difficile burden, toxin B level, and calprotectin level; and postmortem histopathological analysis of colon specimens. Controls were noninfected suckling piglets. Fecal specimens from suckling piglets, formula-fed piglets, and formula-fed, C. difficile-infected piglets were used for metagenomics analysis. High background levels of C. difficile and toxin were detected in formula-fed piglets prior to infection, while suckling piglets carried about 3-fold less C. difficile, and toxin was not detected. Toxin level in C. difficile-challenged animals correlated positively with C. difficile and calprotectin levels. Postmortem signs of CDI were absent in suckling piglets, whereas mesocolonic edema and gas-filled distal small intestines and ceca, cellular damage, and reduced expression of claudins were associated with animals from the challenge trials. Microbiota in formula-fed piglets was enriched with Escherichia, Shigella, Streptococcus, Enterococcus, and Ruminococcus species. Formula-fed piglets were predisposed to C. difficile colonization earlier as compared to suckling piglets. Infection with a hypervirulent C. difficile ribotype did not aggravate the symptoms of infection. Sow-offspring association and consumption of porcine milk during early life may be crucial for the control of C. difficile expansion in piglets.

Hydrostatic pressure incubation affects barrier properties of mammary epithelial cell monolayers, in vitro.

During lactation, accumulation of milk in mammary glands (MG) causes hydrostatic pressure (HP) and concentration of bioactive compounds. Previously, a changed expression of tight junction (TJ) proteins was observed in mice MGs by accumulation of milk, in vivo. The TJ primarily determines the integrity of the MG epithelium. The present study questioned whether HP alone can affect the TJ in a mammary epithelial cell model, in vitro. Therefore, monolayers of HC11, a mammary epithelial cell line, were mounted into modified Ussing chambers and incubated with 10 kPa bilateral HP for 4 h. Short circuit current and transepithelial resistance were recorded and compared to controls, and TJ proteins were analyzed by Western blotting and immunofluorescent staining. In our first approach HC11 cells could withstand the pressure incubation and a downregulation of occludin was observed. In a second approach, using prolactin- and dexamethasone-induced cells, a decrease of short circuit current was observed, beginning after 2 h of incubation. With the addition of 1 mM barium chloride to the bathing solution the decrease could be blocked temporarily. On molecular level an upregulation of ZO-1 could be observed in hormone-induced cells, which was downregulated after the incubation with barium chloride. In conclusion, bilateral HP incubation affects mammary epithelial monolayers, in vitro. Both, the reduction of short circuit current and the change in TJ proteins may be interpreted as physiological requirements for lactation.

Confounding influence of tamoxifen in mouse models of Cre recombinase-induced gene activity or modulation.

Tamoxifen (TAM) is commonly used for cell type specific Cre recombinase-induced gene inactivation and in cell fate tracing studies. Inducing a gene knockout by TAM and using non-TAM exposed mice as controls lead to a situation where differences are interpreted as consequences of the gene knockout but in reality result from TAM-induced changes in hepatic metabolism. The degree to which TAM may compromise the interpretation of animal experiments with inducible gene expression still has to be elucidated. Here, we report that TAM strongly attenuates CCl4-induced hepatotoxicity in male C57Bl/6N mice, even after a 10 days TAM exposure-free period. TAM decreased (p < 0.0001) the necrosis index and the level of aspartate- and alanine transaminases in CCl4-treated compared to vehicle-exposed mice. TAM pretreatment also led to the downregulation of CYP2E1 (p = 0.0045) in mouse liver tissue, and lowered its activity in CYP2E1 expressing HepG2 cell line. Furthermore, TAM increased the level of the antioxidant ascorbate, catalase, SOD2, and methionine, as well as phase II metabolizing enzymes GSTM1 and UGT1A1 in CCl4-treated livers. Finally, we found that TAM increased the presence of resident macrophages and recruitment of immune cells in necrotic areas of the livers as indicated by F4/80 and CD45 staining. In conclusion, we reveal that TAM increases liver resistance to CCl4-induced toxicity. This finding is of high relevance for studies using the tamoxifen-inducible expression system particularly if this system is used in combination with hepatotoxic compounds such as CCl4.

The epithelial barrier and beyond: Claudins as amplifiers of physiological organ functions.

Epithelial cell layers are interconnected by a meshwork of tight junction (TJ) protein strands, which are localized within apicolateral membranes. The proteins that form TJs are regarded to provide a static barrier, determining epithelial properties. However, recent findings in the field of barriology suggest that TJs contribute to more physiological aspects than indicated by the sum of the qualities of the single TJ proteins. Generally, TJs exhibit four major functions: (i) a "gate function," defining transepithelial permeability (i.e., barrier) properties, (ii) a "fence function" determining epithelial cell polarity, (iii) a "signaling function," affecting regulatory pathways, and (iv) a "stabilizing function," maintaining the integrity of the epithelium. This review presents a critical view on how the efficacy of physiological processes in epithelia and thus organ function might be improved by changes in the expression of claudins, the latter representing the largest and most variable family of TJ proteins. Major focus is set on (i) the coordinated regulation of transport and barrier in the intestine, (ii) the role of TJs in defining the route for antigen uptake and presentation in intestinal Peyer's patches, and (iii) the TJ function in mammary glands in response to milk accumulation, which represent impressive examples to highlight the amplification of epithelial functions by TJ proteins. © 2017 IUBMB Life, 69(5):290-296, 2017.

Effects of Ex Vivo Infection with ETEC on Jejunal Barrier Properties and Cytokine Expression in Probiotic-Supplemented Pigs.

BACKGROUND AND AIM: Enterotoxigenic Escherichia coli (ETEC) strains are involved in piglet post-weaning diarrhea. Prophylactic measures including probiotics have been examined in infection experiments with live piglets. In the present study, we have tested whether the early effects of ETEC infection can also be evoked and studied in a model in which ETEC is added to whole mucosal tissues ex vivo, and whether this response can be modulated by prior supplementation of the piglets with probiotics. METHODS: Jejunal barrier and transport properties of Enterococcus faecium-supplemented or control piglets were assessed in Ussing chambers. Part of the epithelia was challenged with an ETEC strain at the mucosal side. Fluxes of fluorescein as a marker of paracellular permeability, and the expression of selected tight junction (TJ) proteins and of proinflammatory cytokines were measured. RESULTS: The addition of ETEC ex vivo induced an increase in transepithelial resistance peaking in the first 2 h with a concomitant reduction in fluorescein fluxes, indicating tightening effects on barrier function. The response of short-circuit current after stimulation with PGE2 or glucose was reduced in epithelia treated with ETEC. ETEC induced a decrease in the TJ protein claudin-4 in the control diet group after 280 min and an increase in the mRNA expression of the proinflammatory cytokines interleukin-8 and TNF-α in both groups after 180 min. CONCLUSIONS: The addition of ETEC ex vivo affected barrier function and transport properties of the jejunal tissues and enhanced cytokine expression. The differences in claudin-4 expression in the jejunum might indicate a beneficial effect of E. faecium prefeeding.

Claudin clusters as determinants of epithelial barrier function.

Claudins are tetraspan tight junction proteins which have been attributed to primarily determine epithelial barrier function in a wide variety of different organs and tissues. Among this protein family with currently 27 members, single claudins contribute in an organ- and tissue-specific manner to defined properties such as cation-, anion- or water-selective pore functions, sealing functions or ambiguous functions. As the size of tight junction strand particles visualized by freeze-fracture electron microscopy have a diameter of approximately 10 nm, multimeric assembly of tight junction proteins appears to be a basic principle for barrier formation. Moreover, expression patterns of different tissues showed that single claudins appear to specifically co-localize with other claudins, which indicates a cluster formation within tight junction strand particles with a fixed stoichiometry. This review provides a critical view on the current understanding of tight junction protein co-localization within strands. We analyze how tissue specific differences of claudin functions could be dependent on their specific partners for barrier formation. Furthermore, a model of claudin clusters as structural and functional units within tight junction strands is provided.

Transient opening of the perineurial barrier for analgesic drug delivery.

Selective targeting of sensory or nociceptive neurons in peripheral nerves remains a clinically desirable goal. Delivery of promising analgesic drugs is often impeded by the perineurium, which functions as a diffusion barrier attributable to tight junctions. We used perineurial injection of hypertonic saline as a tool to open the perineurial barrier transiently in rats and elucidated the molecular action principle in mechanistic detail: Hypertonic saline acts via metalloproteinase 9 (MMP9). The noncatalytic hemopexin domain of MMP9 binds to the low-density lipoprotein receptor-related protein-1, triggers phosphorylation of extracellular signal-regulated kinase 1/2, and induces down-regulation of the barrier-forming tight junction protein claudin-1. Perisciatic injection of any component of this pathway, including MMP9 hemopexin domain or claudin-1 siRNA, enables an opioid peptide ([D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin) and a selective sodium channel (NaV1.7)-blocking toxin (ProToxin-II) to exert antinociceptive effects without motor impairment. The latter, as well as the classic TTX, blocked compound action potentials in isolated nerves only after disruption of the perineurial barrier, which, in return, allowed endoneurally released calcitonin gene-related peptide to pass through the nerve sheaths. Our data establish the function and regulation of claudin-1 in the perineurium as the major sealing component, which could be modulated to facilitate drug delivery or, potentially, reseal the barrier under pathological conditions.