Intestinal Epithelial Cell Layer Research Articles

P1 Interplay of flagellar motility and mucin degradation stimulates the penetration of Pseudomonas aeruginosa through the mucin layer

Introduction:Thehost intestinal epithelial cell layer and extracellular mucusplaya key role inpreventing infection fromthe vast numbers of enteric microorganisms.Pseudomonas aeruginosa can penetrate these barriers and set the stage for the development of invasive infections suchas gut-derived sepsis in immunocompromisedpatients. Wehave demonstrated that a type III effector, ExoS, facilitates P. aeruginosa penetration through the epithelial barrier by impairing the defense of tight junctions. However, how P. aeruginosa penetrates the mucin layer to reach the epithelial cell surface remains largely unknown. Objectives: Here, we found that two efficient mechanisms, flagellar motility and mucin degradation, are needed for penetration of P. aeruginosa through the mucin barrier. Methods: (1) Association assay: Bacterial association was evaluated by human epithelial colorectal adenocarcinoma (Caco-2) cells at 1 h and 2h after infection; (2) Mucin penetration assay: Mucin, from bovine submaxillary glands, was prepared in Transwell®. Bacteria were loaded on the top of the mucin layer, and the numbers of bacteria in the basolateralmediumwere counted at 1 h; and (3)Mucin degradation assay: Following infection, the mucus layer of the Caco-2 cells was stained with the Periodic Acid-Schiff (PAS) Staining and the MUC2 protein was analyzed by immunoblot with anti-MUC2 antibody. Results: Deletion of the flagellar motility-related gene, the filament protein gene fliC and the motor complex protein genes motABCD , from P. aeruginosa PAO1 decreased association of P. aeruginosa with the apical surface of Caco-2 cells and penetration to an artificial mucin layer. Additionally, the presence of P. aeruginosa decreased the total mucin, including the secreted mucin protein MUC2 on the surface of the Caco-2 cell monolayer, regardless of flagellar motility. Construction of the PAO1 mutant series knockedout 12 putative serine protease genes and identified the mucD gene, which participated in degradation of total mucin including MUC2. Furthermore, decreased association with the Caco-2 cell monolayer was observed in the mucD mutant, and the decrease was synergistically amplified by double knock-out with fliC . Conclusion: P. aeruginosa can penetrate the mucin layer using flagellar motility and mucin degradation, which is dependent on the MucD protease or the mucD related-gene protease. This information, in addition to further analysis of the mechanisms of P. aeruginosa penetration of the epithelial cell layer followingmucin layer penetration, will lead to a greater understanding of P. aeruginosa infection via translocation in the human intestinal tract. P2 Genetic research of leprosy F. Zhang1 *, H. Liu1, S. Chen1, J. Liu2. 1 Shandong Provincial Institute of Dermatology and Venereology, Jinan, China , 2 Human Genetics, Genome Institute of Singapore, Singapore, Singapore E-mail address : hongyue2519@hotmail.com

Using a continuum model to predict closure time of gaps in intestinal epithelial cell layers

A two-dimensional continuum model of collective cell migration is used to predict the closure of gaps in intestinal epithelial cell layers. The model assumes that cell migration is governed by lamellipodia formation, cell-cell adhesion, and cell-substrate adhesion. Model predictions of the gap edge position and complete gap closure time are compared with experimental measures from cell layer scratch assays (also called scratch wound assays). The goal of the study is to combine experimental observations with mathematical descriptions of cell motion to identify effects of gap shape and area on closure time and to propose a method that uses a simple measure (e.g., area) to predict overall gap closure time early in the closure process. Gap closure time is shown to increase linearly with increasing gap area; however, gaps of equal areas but different aspect ratios differ greatly in healing time. Previous methods that calculate overall healing time according to the absolute or percent change in gap area assume that the gap area changes at a constant rate and typically underestimate gap closure time. In this study, data from scratch assays suggest that the rate of change of area is proportional to the first power or square root power of area.

A mouse model of pathological small intestinal epithelial cell apoptosis and shedding induced by systemic administration of lipopolysaccharide

SUMMARYThe gut barrier, composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, prevents the entrance of harmful microorganisms, antigens and toxins from the gut lumen into the blood. Small intestinal homeostasis is normally maintained by the rate of shedding of senescent enterocytes from the villus tip exactly matching the rate of generation of new cells in the crypt. However, in various localized and systemic inflammatory conditions, intestinal homeostasis can be disturbed as a result of increased IEC shedding. Such pathological IEC shedding can cause transient gaps to develop in the epithelial barrier and result in increased intestinal permeability. Although pathological IEC shedding has been implicated in the pathogenesis of conditions such as inflammatory bowel disease, our understanding of the underlying mechanisms remains limited. We have therefore developed a murine model to study this phenomenon, because IEC shedding in this species is morphologically analogous to humans. IEC shedding was induced by systemic lipopolysaccharide (LPS) administration in wild-type C57BL/6 mice, and in mice deficient in TNF-receptor 1 (Tnfr1−/−), Tnfr2 (Tnfr2−/−), nuclear factor kappa B1 (Nfκb1−/−) or Nfĸb2 (Nfĸb2−/−). Apoptosis and cell shedding was quantified using immunohistochemistry for active caspase-3, and gut-to-circulation permeability was assessed by measuring plasma fluorescence following fluorescein-isothiocyanate–dextran gavage. LPS, at doses ≥0.125 mg/kg body weight, induced rapid villus IEC apoptosis, with peak cell shedding occurring at 1.5 hours after treatment. This coincided with significant villus shortening, fluid exudation into the gut lumen and diarrhea. A significant increase in gut-to-circulation permeability was observed at 5 hours. TNFR1 was essential for LPS-induced IEC apoptosis and shedding, and the fate of the IECs was also dependent on NFκB, with signaling via NFκB1 favoring cell survival and via NFκB2 favoring apoptosis. This model will enable investigation of the importance and regulation of pathological IEC apoptosis and cell shedding in various diseases.

Interplay of flagellar motility and mucin degradation stimulates the uassociation of Pseudomonas aeruginosa with human epithelial colorectal adenocarcinoma (Caco-2) cells

Pseudomonas aeruginosa can penetrate the extracellular mucin barrier formed by the intestinal epithelial cell layer and establish gut-derived sepsis in immunocompromised patients. We found that two efficient mechanisms, flagellar motility and mucin degradation, are needed for penetration of P. aeruginosa through the mucin barrier. Deletion of the flagellar motility-related gene, the filament protein gene fliC, the cap protein gene fliD, and the motor complex protein genes motABCD from P. aeruginosa PAO1 decreased association of P. aeruginosa with the apical surface of human epithelial colorectal adenocarcinoma (Caco-2) cells. A penetration experiment using an artificial mucin layer suggested that the decreased penetration is caused by attenuation of mucin penetration ability. Additionally, the presence of P. aeruginosa decreased the total mucin, including the secreted mucin protein MUC2, on the surface of the Caco-2 cell monolayer, regardless of flagellar motility. Construction of the PAO1 mutant series knocked out 12 putative serine protease genes and identified the mucD gene, which participated in degradation of total mucin, including MUC2. Furthermore, decreased association with the surface of the Caco-2 cell monolayer was observed in the mucD mutant, and the decrease was synergistically amplified by double knockout with fliC. We conclude that P. aeruginosa can penetrate the mucin layer using flagellar motility and mucin degradation, which is dependent on the MucD protease or the mucD gene-related protease.

Gene Regulation of Intestinal Porcine Epithelial Cells IPEC-J2 Is Dependent on the Site of Deoxynivalenol Toxicological Action

The intestinal epithelial cell layer represents the border between the luminal and systemic side of the gut. The decision between absorption and exclusion of substances is the quintessential function of the gut and varies along the gut axis. Consequently, potentially toxic substances may reach the basolateral domain of the epithelial cell layer via blood stream. The mycotoxin deoxynivalenol (DON) is a Fusarium derived secondary metabolite known to enter the blood stream and displaying a striking toxicity on the basolateral side of polarised epithelial cell layers in vitro. Here we analysed potential mechanisms of apical and basolateral DON toxicity reflected in the gene expression. We used the jejunum-derived, polarised intestinal porcine epithelial cell line IPEC-J2 as an in vitro cell culture model. Luminal and systemic DON challenge of the epithelial cell layer was mimicked by a DON application from the apical or basolateral compartment of membrane inserts for 72 h. We compared the genome-wide gene expression of untreated and DON-treated IPEC-J2 cells with the GeneChip® Porcine Genome Array of Affymetrix. Low basolateral DON (200 ng/mL) application triggered 10 times more gene transcripts in comparison to the corresponding apical application (2539 versus 267) despite the intactness of the challenged cell layer as measured by transepithelial electrical resistance. Analysis of the regulated genes by bioinformatic resource DAVID identified several groups of biochemical pathways modulated by concentration and orientation of DON application. Selected genes representing pathways of the cellular metabolism, information processing and structural design were analysed in detail by quantitative PCR. Our findings clearly show that apical and basolateral challenge of epithelial cell layers trigger different gene response profiles paralleled with a higher susceptibility towards basolateral challenge. The evaluation of toxicological potentials of mycotoxins should take this difference in gene regulation dependent on route of application into account.

Toll‐like receptor 5 (TLR5), but not TLR4 or 3, induces TNFα production by intestinal epithelial cells (IEC), which is enhanced by suppressor of cytokine signalling 3 (SOCS3)

A single layer of IEC, expressing TLR, line the intestine. TLR‐induced inflammatory pathways are vital for mucosal homeostasis and dysregulated repair may predispose to inflammatory bowel disease (IBD). In IBD, the expression of SOCS3, a negative feedback inhibitor of the JAK/STAT pathway, is enhanced. The aim of this study was to establish the function of SOCS3 in TLR‐mediated epithelial homeostasis. We developed SW480 IEC cell lines to over‐express SOCS3 (SOCS3hi), to study gene expression, vs. SOCS3norm IEC in response to microbial stimulation. IEC production of pro‐inflammatory TNFα mRNA was upregulated following stimulation with flagellin (TLR5), but not LPS (TLR4) or Poly I:C (TLR3), at varying concentrations. This response was significantly enhanced in SOCS3hi vs. SOCS3normIEC: 31.9 ± 10 vs. 8.7 ± 1.8 fold increase at 1ug/ml (p≤0.05), 20.3 ± 4.4 vs. 6.4 ± 1.6 fold‐increase at 0.1ug/ml (p≤0.03) and 15.1 ± 5.1 vs. 4 ± 1.1 fold‐increase at 0.01ug/ml (p≤0.05), compared with no‐treatment control mRNA levels. Our studies indicate that SOCS3 is not only an inhibitor of epithelial repair, but may help to perpetuate the inflammatory response by promoting the production of pro‐inflammatory TNFα.

Intestinal Epithelial Cells with Impaired Autophagy Lose Their Adhesive Capacity in the Presence of TNF-α

Genome-wide association studies have revealed a link between autophagy-related (ATG) genes and susceptibility to Crohn's disease. This suggests underlying involvement of autophagy impairment in the pathogenesis of Crohn's disease. This study was performed to investigate the pathophysiological importance of autophagy impairment in intestinal epithelial cells exposed to TNF-α. Human colonic epithelial cells (HT-29) and rat small intestinal epithelial cells (IEC-18) were used. Formation of phosphatidylethanolamine-conjugated microtubule-associated protein light chain 3 (LC3-II) was monitored as a marker of autophagy. Autophagy was inhibited using 3-methyladenine or short interfering RNA targeting ATG5 and ATG16L1. TNF-α treatment elicited a significant dose-dependent increase in LC3-II protein levels, thus autophagy is induced in the presence of TNF-α. Combined autophagy inhibition and TNF-α treatment induced a marked increase in the number of detached cells and a decrease in activated integrin β1 protein levels. Trypan blue staining indicated 70-80 % of the detached cells were alive, suggesting that these cells became detached not because they were killed but because of dysfunction of cellular adhesion. This is the first study indicating that intestinal epithelial cells with impaired autophagy lose their adhesive capacity in the presence of TNF-α. These observations indicate that impairment of autophagy leads to disruption of the intestinal epithelial cell layers in TNF-α-rich environments.

Role of suppressor of cytokine signalling 3 in regulating microbe‐mediated intestinal epithelial proliferation and repair

The gut is lined with a single layer of intestinal epithelial cells (IEC) that functions as a barrier between a plethora of luminal antigens, including commensal bacteria, and a primed immune system. In a healthy adult, the entire epithelium is replaced about every 5 days; therefore maintenance of the IEC barrier is essential in maintaining mucosal homeostasis. IEC express Toll-like receptors (TLR) which recognise microbial ligands and recent evidence proposes that IEC homeostasis and repair are mediated through TLR-induced inflammatory pathways. Resistance to helminthic infection relies on hyperproliferation of IEC to eliminate the parasites. Suppressor of cytokine signalling 3 (SOCS3) is a natural endogenous modulator of epithelial turnover which limits hyperproliferation of aberrant IEC predisposing to dysplasia and cancer. We have used model human SW480 and Caco2 IEC cell lines to study IEC proliferation and wound healing responses to lipopolysaccharide (LPS; 1μg/ml)and Trichuris muris secreted protein (ES; 5 μg/ml); to determine their effects on epithelial proliferation and repair and define the role of SOCS3 in modulating microbe-mediated IEC homeostasis. Exposure to LPS and ES significantly enhanced SW480 proliferation compared with no treatment controls after 48h culture by 12 ± 1.6% and 21 ± 6.5% respectively. Increased wound healing in differentiated Caco2 monolayers was observed at 6h following treatment with LPS and ES, but this increased was not observed at 24, 48 or 72h compared with no treatment controls. IEC over-expression of SOCS3 inhibited microbe-induced increases in proliferation (LPS 17 ± 4%; ES 13 ± 3.5%), but did not significantly change wound healing responses. These findings suggest that although SOCS3 may limit microbe-induced proliferation of IEC, SOCS3 does not inhibit initial epithelial wound healing responses.

A Novel Role for CD46 in Wound Repair

The intestinal epithelium not only provides a vital physical barrier between the host and environment but is also required for uptake of nutrients and the induction of tolerance against commensals. Deregulation of any of these functions leads to several disease states including chronic infection, inflammatory bowel disease, and cancer. Here, we describe a novel role for the complement regulator CD46 in the regulation of intestinal epithelial cell (IEC) barrier function. We found that CD46 directly interacts in IECs with the cytoplasmic kinase SPAK and with transmembrane E-cadherin, both proteins necessary for epithelial cell junction and barrier formation. Further, CD46 activation on Caco-2 cells induced rapid and significant decrease in transepithelial resistance with concomitant increase in paracellular permeability. Importantly, though CD46 activation of IEC layers allowed for increased transgression of pathogenic E. coli, it also increased epithelial cell proliferation and accelerated wound repair. These data suggest a previously unappreciated role for CD46 in the maintenance of epithelial cell barrier integrity as well as barrier repair. However, this role for CD46 as “gate keeper” of the epithelium could also provide reason as to why so many pathogens bind to CD46 as such event would facilitate infection.

Potentiation of Epithelial Innate Host Responses by Intercellular Communication

The epithelium efficiently attracts immune cells upon infection despite the low number of pathogenic microbes and moderate levels of secreted chemokines per cell. Here we examined whether horizontal intercellular communication between cells may contribute to a coordinated response of the epithelium. Listeria monocytogenes infection, transfection, and microinjection of individual cells within a polarized intestinal epithelial cell layer were performed and activation was determined at the single cell level by fluorescence microscopy and flow cytometry. Surprisingly, chemokine production after L. monocytogenes infection was primarily observed in non-infected epithelial cells despite invasion-dependent cell activation. Whereas horizontal communication was independent of gap junction formation, cytokine secretion, ion fluxes, or nitric oxide synthesis, NADPH oxidase (Nox) 4-dependent oxygen radical formation was required and sufficient to induce indirect epithelial cell activation. This is the first report to describe epithelial cell-cell communication in response to innate immune activation. Epithelial communication facilitates a coordinated infectious host defence at the very early stage of microbial infection.

N-cadherin can structurally substitute for E-cadherin during intestinal development but leads to polyp formation

We conditionally substituted E-cadherin (E-cad; cadherin 1) with N-cadherin (N-cad; cadherin 2) during intestine development by generating mice in which an Ncad cDNA was knocked into the Ecad locus. Mutant mice were born, demonstrating that N-cad can structurally replace E-cad and establish proper organ architecture. After birth, mutant mice gradually developed a mutant phenotype in both the small and large intestine and died at ~2-3 weeks of age, probably due to malnutrition during the transition to solid food. Molecular analysis revealed an extended domain of cells from the crypt into the villus region, with nuclear localization of β-catenin (β-cat; Ctnnb1) and enhanced expression of several β-cat target genes. In addition, the BMP signaling pathway was suppressed in the intestinal epithelium of the villi, suggesting that N-cad might interfere with BMP signaling in the intestinal epithelial cell layer. Interestingly, mutant mice developed severe dysplasia and clusters of cells with neoplastic features scattered along the crypt-villus axis in the small and large intestine. Our experimental model indicates that, in the absence of E-cad, the sole expression of N-cad in an epithelial environment is sufficient to induce neoplastic transformations.

N-cadherin can structurally substitute for E-cadherin during intestinal development but leads to polyp formation

We conditionally substituted E-cadherin (E-cad; cadherin 1) with N-cadherin (N-cad; cadherin 2) during intestine development by generating mice in which an Ncad cDNA was knocked into the Ecad locus. Mutant mice were born, demonstrating that N-cad can structurally replace E-cad and establish proper organ architecture. After birth, mutant mice gradually developed a mutant phenotype in both the small and large intestine and died at ~2-3 weeks of age, probably due to malnutrition during the transition to solid food. Molecular analysis revealed an extended domain of cells from the crypt into the villus region, with nuclear localization of beta-catenin (beta-cat; Ctnnb1) and enhanced expression of several beta-cat target genes. In addition, the BMP signaling pathway was suppressed in the intestinal epithelium of the villi, suggesting that N-cad might interfere with BMP signaling in the intestinal epithelial cell layer. Interestingly, mutant mice developed severe dysplasia and clusters of cells with neoplastic features scattered along the crypt-villus axis in the small and large intestine. Our experimental model indicates that, in the absence of E-cad, the sole expression of N-cad in an epithelial environment is sufficient to induce neoplastic transformations.

Rapid detachment of Giardia lamblia trophozoites as a mechanism of antimicrobial action of the isoflavone formononetin

Attachment to the small intestinal mucosa is crucial for initiating and maintaining Giardia infection. We tested the effect of isoflavones on Giardia attachment. We evaluated the effect of formononetin on trophozoite attachment to glass, to intestinal epithelial cell layers in vitro and to murine small intestinal explants, and on the intestinal load in mice. We found that the isoflavone formononetin inhibits both attachment and flagellar motility within minutes and reduces the trophozoite load of Giardia in mice within 1.5 h after treatment. The antigiardial activity of formononetin is at least partially due to its capacity to rapidly detach trophozoites.

Involvement of Sialic Acid in Transport of Serotype C1 Botulinum Toxins through Rat Intestinal Epithelial Cells

Clostridium botulinum produces a large toxin complex (L-TC) composed of neurotoxin (BoNT) and non-toxic proteins. In animal botulism, BoNT or L-TC is absorbed via the intestinal epithelium. To establish the cellular mechanisms of botulinum toxin absorption, we used cultured rat intestinal epithelial cells to test the binding and transport of serotype C1 BoNT and L-TC through the cell layers. BoNT and L-TC bound to and passed through the cell layers, with L-TC exhibiting larger binding and transport. Binding and transport of these toxins were inhibited by N-acetyl neuraminic acid or neuraminidase treatment of the cells. These results suggest that binding of serotype C1 BoNT and L-TC to sialic acid on the cells promoted their transport through intestinal epithelial cell layers.

Continuum Elastic Model of Epithelial Sheet Migration

A key component of a wound healing process is rapid migration of epithelial cells that covers the wound area and helps to protect the underlying tissue. In our recent paper∗ we have developed a one-dimensional continuum mechanical model of intestinal epithelial cell layer, that incorporates lamellipod forces at both the wound edge and in the interior of the layer, adhesion forces between the cell layer and the substrate, and elastic stress within the cell layer. Here, the model is extended to two-dimensions and solved numerically using a level set method, which tracks the moving wound edge on a fixed grid. The model is calibrated by comparing the position of wound edge with experimentally observed positions in scratch-wound assay experiments. These comparisons show good qualitative agreement between model results and experimental observations. The models supports experimental observations that the time to wound closure varies with initial wound shape and area, and that the closure is possible, albeit slower, if boundary lamellipod formation is inhibited.∗ Qi. et al., One-dimensional elastic continuum model of enterocyte layer migration, Biohys. J., 93, 3745-3752 (2007).

Transepithelial activation of human leukocytes by probiotics and commensal bacteria: Role of Enterobacteriaceae‐type endotoxin

The goal of the current study was to clarify whether commercially available probiotics induce greater trans-epithelial activation of human leukocytes than do commensal, food-derived and pathogenic bacteria and to identify the compounds responsible for this activation. Eleven different bacterial strains, and some of their pathogen-associated molecular patterns, were incubated apically on a confluent layer of intestinal epithelial cells (Caco-2), which were basolaterally co-cultured with human mononuclear leukocytes. Only Gram-negative bacteria having Enterobacteriaceae-type endotoxin (commensal Escherichia coli K12, probiotic E. coli Nissle, EPEC) induced basolateral production of TNF-alpha, IFN-gamma, IL 6, 8, and 10. Gram-positive probiotics (Lactobacillus spp. and Bifidobacterium spp.) had virtually no effect. In addition, commensals (Enterococcus faecalis, Bacteroides vulgatus) and food fermenters (Lactobacillus spp.) did not stimulate leukocytes transepithelially. Endotoxin from E. coli and Salmonella enterica Typhimurium induced comparable transepithelial stimulation of leukocytes, but not endotoxin from B. vulgatus or lipoteichoic acid from E. faecalis. Endotoxin-binding agents (polymyxin, colistin) completely abrogated transepithelial activation of leukocytes. Enterobacteriaceae-type endotoxin is a crucial factor in transepithelial stimulation of leukocytes, regardless of whether it is produced by probiotics or other bacteria. Hence, transepithelial stimulation ofleukocytes' innate immune response seems to not be linked to the health-promoting effects ofprobiotics.

Role of the intestinal barrier in inflammatory bowel disease

A critical function of the intestinal mucosa is to form a barrier that separates luminal contents from the interstitium. The single layer of intestinal epithelial cells (IECs) serves as a dynamic interface between the host and its environment. Cell polarity and structural properties of the epithelium is complex and is important in the development of epithelial barrier function. Epithelial cells associate with each other via a series of intercellular junctions. The apical most intercellular junctional complex referred to as the Apical Junction Complex (AJC) is important in not only cell-cell recognition, but also in the regulation of paracellular movement of fluid and solutes. Defects in the intestinal epithelial barrier function have been observed in a number of intestinal disorders such as inflammatory bowel disease (IBD). It is now becoming evident that an aberrant epithelial barrier function plays a central role in the pathophysiology of IBD. Thus, a better understanding of the intestinal epithelial barrier structure and function in healthy and disease states such as IBD will foster new ideas for the development of therapies for such chronic disorders.

Intestinal Drug Absorption Enhancers: Synergistic Effects of Combinations

Although many absorption enhancers have been investigated, very few are used clinically. A need exists therefore for more effective absorption enhancers. The drug-absorption-enhancing effects of combinations of N-trimethyl chitosan chloride (TMC) with degrees of quaternization of 48 and 64%, dicarboxymethyl chitosan oligosaccharide, and chitosan lactate oligomer with monocaprin and melittin were compared to their individual performances using the in vitro Caco-2 cell model. Combining the absorption enhancers showed synergism in both the reduction of the transepithelial electrical resistance (TEER) and the enhancement of the transport of a macromolecular model compound across this intestinal epithelial cell layer. Lower concentrations of the absorption enhancers in the combination groups exhibited greater effects on the epithelial cells compared with the individual absorption enhancers.

Conjugated primary bile salts reduce permeability of endotoxin through intestinal epithelial cells and synergize with phosphatidylcholine in suppression of inflammatory cytokine production

Endotoxemia was shown to be integral in the pathophysiology of obstructive jaundice. In the current study, the role of conjugated primary bile salts (CPBS) and phosphatidylcholine on the permeability of endotoxin through a layer of intestinal epithelial cells and the consequent activation of basolaterally cocultured human mononuclear leukocytes were measured. In a coculture model, a layer of differentiated, confluent Caco-2 cells was apically stimulated with growth-arrested, nonpathogenic Escherichia coli. Basic human cell culture laboratory. The effect of CPBS (0.5 mM and 1.5 mM), phosphatidylcholine (0.38 mM), and human bile (0.5% vol/vol) on the barrier function was assessed by the measurement of transepithelial electrical resistance, by endotoxin permeability through the intestinal epithelial cell layer, and by basolateral cytokine enzyme-linked immunosorbent assay measurement (tumor necrosis factor-[alpha], interleukins-6, -8, and -10). Micelles formed by CPBS were detected by dynamic light scattering. The association of endotoxin with CPBS micelles was tested by fluorescence resonance energy transfer. Apical addition of CPBS suppressed the permeability of endotoxins through the intestinal epithelial cell layer significantly. In parallel, apical supplementation of CPBS dose-dependently reduced the basolateral production of all cytokines measured. Apical phosphatidylcholine supplementation enhanced this effect significantly. CPBS formed micelles (diameter, 134 +/- 7 nm), which were able to bind endotoxin to their surface. CPBS can reduce the permeation of endotoxin through intestinal epithelial cell layers by binding it to micelles. Thereby, the inflammatory processes beyond the mucosal surface are suppressed, an effect that is enhanced by phosphatidylcholine.

Epithelial Cell IκB-Kinase β Has an Important Protective Role inClostridium difficileToxin A-Induced Mucosal Injury

Toxin A released by Clostridium difficile interacts with the single layer of intestinal epithelial cells that lines the host's intestinal tract and leads to mucosal damage and inflammation that manifests clinically as antibiotic-associated diarrhea and pseudomembranous colitis. Activation of the transcription factor NF-kappaB in intestinal epithelial cells is important for regulating the expression of epithelial cell proinflammatory genes and cell survival. However, the role of NF-kappaB activation in the pathogenesis of C. difficile toxin A-induced colitis is unknown. To determine the functional importance in vivo of NF-kappaB activation in intestinal epithelium in the pathogenesis of C. difficile-induced colitis, we used mutant mice that do not activate the classical NF-kappaB signaling pathway in intestinal epithelial cells due to a conditional deficiency in those cells of the IkappaB-kinase beta (IKKbeta) subunit of IKK. C. difficile toxin A challenge of intestinal loops in intestinal epithelial cell IKKbeta-deficient mice induced a rapid and significant increase in intestinal epithelial apoptosis compared with littermate controls. This was accompanied by a significant increase in acute mucosal inflammation, mucosal injury, luminal fluid secretion, and bacterial translocation. We conclude that activation of intestinal epithelial cell NF-kappaB by toxin A plays an important host mucosal protective role after C. difficile toxin A exposure that is mediated, at least in part, through promoting epithelial cell survival by abrogating epithelial cell apoptosis.