Uptake of Tropheryma whipplei by Intestinal Epithelia.

BACKGROUND: Tropheryma whipplei (TW) can cause different pathologies, e.g., Whipple's disease and transient gastroenteritis. The mechanism by which the bacteria pass the intestinal epithelial barrier, and the mechanism of TW-induced gastroenteritis are currently unknown. METHODS: Using ex vivo disease models comprising human duodenal mucosa exposed to TW in Ussing chambers, various intestinal epithelial cell (IEC) cultures exposed to TW and a macrophage/IEC coculture model served to characterize endocytic uptake mechanisms and barrier function. RESULTS: TW exposed ex vivo to human small intestinal mucosae is capable of autonomously entering IECs, thereby invading the mucosa. Using dominant-negative mutants, TW uptake was shown to be dynamin- and caveolin-dependent but independent of clathrin-mediated endocytosis. Complementary inhibitor experiments suggested a role for the activation of the Ras/Rac1 pathway and actin polymerization. TW-invaded IECs underwent apoptosis, thereby causing an epithelial barrier defect, and were subsequently subject to phagocytosis by macrophages. CONCLUSIONS: TW enters epithelia via an actin-, dynamin-, caveolin-, and Ras-Rac1-dependent endocytosis mechanism and consecutively causes IEC apoptosis primarily in IECs invaded by multiple TW bacteria. This results in a barrier leak. Moreover, we propose that TW-packed IECs can be subject to phagocytic uptake by macrophages, thereby opening a potential entry point of TW into intestinal macrophages.

Tricellulin Effect on Paracellular Water Transport.

In epithelia, large amounts of water pass by transcellular and paracellular pathways, driven by the osmotic gradient built up by the movement of solutes. The transcellular pathway has been molecularly characterized by the discovery of aquaporin membrane channels. Unlike this, the existence of a paracellular pathway for water through the tight junctions (TJ) was discussed controversially for many years until two molecular components of paracellular water transport, claudin-2 and claudin-15, were identified. A main protein of the tricellular TJ (tTJ), tricellulin, was shown to be downregulated in ulcerative colitis leading to increased permeability to macromolecules. Whether or not tricellulin also regulates water transport is unknown yet. To this end, an epithelial cell line featuring properties of a tight epithelium, Madin-Darby canine kidney cells clone 7 (MDCK C7), was stably transfected with small hairpin RNA (shRNA) targeting tricellulin, a protein of the tTJ essential for the barrier against passage of solutes up to 10 kDa. Water flux was induced by osmotic gradients using mannitol or 4 and 40 kDa-dextran. Water flux in tricellulin knockdown (KD) cells was higher compared to that of vector controls, indicating a direct role of tricellulin in regulating water permeability in a tight epithelial cell line. We conclude that tricellulin increases water permeability at reduced expression.

Claudin-related intestinal diseases.

With up to 200 m(2) the human intestine is the organ with the largest absorptive surface of the body. It is lined by a single layer of epithelial cells that separates the host from the environment. The intestinal epithelium provides both, selective absorption of nutrients, ions, and water but also a highly effective barrier function which includes the first line of defense against environmental antigens. The paracellular part of this barrier function is provided by tight junction (TJ) proteins, especially the large family of claudins. Changes in abundance or molecular structure of claudins can generally result in three typical effects, (i) decreased absorptive passage, (ii) increased secretory passage of small solutes and water causing leak flux diarrhea and (iii) increased absorptive passage of macromolecules which may induce inflammatory processes. Several intestinal diseases are associated with such changes that can result in intestinal inflammation and symptoms like weight loss, abdominal pain or diarrhea. This review summarizes our current knowledge on barrier dysfunction and claudin dysregulation in several intestinal diseases gastroenterologists are often faced with, like inflammatory bowel disease, microscopic colitis, celiac disease, irritable bowel syndrome, gallstones and infectious diseases like HIV enteropathy, Campylobacter jejuni and Clostridium perfringens infection.

Zinc strengthens the jejunal barrier by reversibly tightening the paracellular route.

During the postweaning period, piglets are prone to gastrointestinal infections. The resulting impairment of intestinal barrier function may cause diarrhea associated with growth retardation or even death of piglets. Orally applied Zn is commonly used to prevent and treat diarrhea, but its mode of action still needs to be elucidated. To analyze the molecular mechanism whereby Zn acts on porcine intestinal barrier function, ex vivo studies on piglet jejunum and accompanying in vitro studies on a porcine jejunal epithelial cell line, IPEC-J2/PS, were performed with electrophysiological tools. Feeding pharmacological Zn doses exerted no significant electrophysiologically ascertainable short- and long-term effects on jejunal barrier function ex vivo. However, in IPEC-J2/PS, basolateral Zn was cytotoxic since its application caused a release of lactate dehydrogenase and an irreversible breakdown of the epithelial barrier. In contrast, apical Zn application caused an immediate increase in paracellular resistance and a decrease in permeability to the paracellular marker fluorescein, reflecting overall barrier strengthening in vitro. Apical effects were fully reversible upon washout. This indicates that Zn supplemented to feed was completely washed out during ex vivo jejunum preparation. We conclude that there is no evidence for long-term barrier effects through prophylactic Zn supplementation and that extracellular Zn acts acutely and reversibly from the apical side via tightening the paracellular route, thus counteracting leak-flux diarrhea.NEW & NOTEWORTHY Therapeutically administered Zn successfully treats diarrhea in veterinary and human medicine. Here we present data that Zn strengthens the porcine jejunal epithelial barrier by reversibly tightening the paracellular route for inorganic ions and small solutes. Acute or long-lasting Zn effects on transcellular transport (Cl- secretion) were not detected. We therefore conclude that Zn is useful for acutely treating leak-flux diarrhea rather than secretory diarrhea. Suitability as prophylactic feed supplement, however, is questionable.

Yersinia enterocolitica Affects Intestinal Barrier Function in the Colon.

Infection with Yersinia enterocolitica causes acute diarrhea in early childhood. A mouse infection model presents new findings on pathological mechanisms in the colon. Symptoms involve diarrhea with watery feces and weight loss that have their functional correlates in decreased transepithelial electrical resistance and increased fluorescein permeability. Y. enterocolitica was present within the murine mucosa of both ileum and colon. Here, the bacterial insult was of focal nature and led to changes in tight junction protein expression and architecture. These findings are in concordance with observations from former cell culture studies and suggest a leak flux mechanism of diarrhea.

Tight junction, selective permeability, and related diseases.

The tight junction forms a barrier against unlimited paracellular passage but some of the tight junction proteins just do the opposite, they form extracellular channels zigzagging between lateral membranes of neighboring cells. All of these channel-forming proteins and even some of the barrier formers exhibit selectivity, which means that they prefer certain substances over others. All channel formers exhibit at least one of the three types of selectivity: for cations (claudin-2, -10b, -15), for anions (claudin-10a, -17) or for water (claudin-2). Also some, but not all, barrier-forming claudins are charge-selective (claudin-4, -8, -14). Moreover, occludin and tricellulin turned out to be relevant for barrier formation against macromolecule passage. Tight junction proteins are dysregulated or can be genetically defective in numerous diseases, which may lead to three effects: (i) impaired paracellular transport e.g. causing magnesium loss in the kidney, (ii) increased paracellular transport of solutes and water e.g. causing leak-flux diarrhea in the intestine, and (iii) increased permeability to large molecules e.g. unwanted intestinal pathogen uptake fueling inflammatory processes. This review gives an overview on the properties of tight junction proteins featuring selective permeability, and in this context explains how these proteins induce or aggravate diseases.

Hereditary barrier-related diseases involving the tight junction: lessons from skin and intestine.

The tight junction (TJ) regulates paracellular barrier properties. TJs are composed of transmembrane proteins, i.e., claudins, occludin, tricellulin and junctional adhesion molecules as well as TJ plaque proteins. Their relative abundance and composition determines epithelial tightness. TJs undergo rapid regulation by various signalling pathways, either directly addressing TJ transmembrane proteins or via plaque proteins and the cytoskeleton. In the skin, TJs exert predominantly barrier functions, while in the intestine they also mediate paracellular ion and water transport. In diseases, TJs can either be primarily affected (hereditary TJ defects) or changes can result from secondary regulatory inputs as, e.g., in inflammatory diseases (secondary TJ defects). Secondary TJ defects can maintain disease activity, e.g., by enhanced antigen leak. This review discusses TJ composition, function and regulation as well as primary and secondary tight junction defects in a comparative manner in skin and intestine in order to elucidate similarities and differences.

Claudin-17 forms tight junction channels with distinct anion selectivity.

Barrier properties of tight junctions are determined by the claudin protein family. Many claudins seal this barrier, but others form paracellular channels. Among these, no claudins with general and clear-cut anion selectivity have yet been described, while for claudin-10a and claudin-4, only circumstantial or small anion selectivities have been shown. A claudin with unknown function and tissue distribution is claudin-17. We characterized claudin-17 by overexpression and knock-down in two renal cell lines. Overexpression in MDCK C7 cell layers caused a threefold increase in paracellular anion permeability and switched these cells from cation- to anion-selective. Knockdown in LLC-PK(1) cells indorsed the finding of claudin-17-based anion channels. Mutagenesis revealed that claudin-17 anion selectivity critically depends on a positive charge at position 65. Claudin-17 expression was found in two organs: marginal in brain but abundant in kidney, where expression was intense in proximal tubules and gradually decreased towards distal segments. As claudin-17 is predominantly expressed in proximal nephrons, which exhibit substantial, though molecularly not defined, paracellular chloride reabsorption, we suggest that claudin-17 has a unique physiological function in this process. In conclusion, claudin-17 forms channels within tight junctions with distinct anion preference.

Human duodenal organoid-derived monolayers serve as a suitable barrier model for duodenal tissue.

Usually, duodenal barriers are investigated using intestinal cell lines like Caco-2, which in contrast to native tissue are limited in cell-type representation. Organoids can consist of all intestinal cell types and are supposed to better reflect the in vivo situation. Growing three-dimensionally, with the apical side facing the lumen, application of typical physiological techniques to analyze the barrier is difficult. Organoid-derived monolayers (ODMs) were developed to overcome this. After optimizing culturing conditions, ODMs were characterized and compared to Caco-2 and duodenal tissue. Tight junction composition and appearance were analyzed, and electrophysiological barrier properties, like paracellular and transcellular barrier function and macromolecule permeability, were evaluated. Furthermore, transcriptomic data were analyzed. ODMs had tight junction protein expression and paracellular barrier properties much more resembling the originating tissue than Caco-2. Transcellular barrier was similar between ODMs and native tissue but was increased in Caco-2. Transcriptomic data showed that Caco-2 expressed fewer solute carriers than ODMs and native tissue. In conclusion, while Caco-2 cells differ mostly in transcellular properties, ODMs reflect trans- and paracellular properties of the originating tissue. If cultured under optimized conditions, ODMs possess reproducible functionality, and the variety of different cell types makes them a suitable model for human tissue-specific investigations.

Yersinia enterocolitica induces epithelial barrier dysfunction through regional tight junction changes in colonic HT-29/B6 cell monolayers.

Yersinia enterocolitica is a common cause of acute gastroenteritis. This study aimed to clarify the mechanisms leading to barrier dysfunction and diarrhea. Exposure of human colonic HT-29/B6 cells to Y. enterocolitica resulted in a decrease in transepithelial resistance from 404±23 to 163±21 Ω cm² (P<0.001) in parallel with an increase in mannitol (182 Da) and fluorescein (332 Da) permeability, whereas short circuit current did not change. This effect was time dependent, required the presence of living bacteria, could not be triggered by bacterial supernatants and was not due to Yersinia outer proteins. Concomitantly, Y. enterocolitica induced necrosis as indicated by an increase in lactate dehydrogenase-release, whereas epithelial apoptosis was not upregulated. Local changes in conductivity were detected by conductance scanning, indicating 'leaky regions' within the epithelium that were visualized by biotinylation and confocal microscopy. In these regions, claudin-3 and -4 and, especially claudin-8, were redistributed off the tight junction (TJ) into the cytoplasm. In addition, the expression of claudin-2, -3, -8, -10 and ZO-1 was diminished as quantified by immunoblotting. Moreover, we found claudin-8 to be regulated by the c-Jun N-terminal kinase, the inhibition of which attenuated the Y. enterocolitica-induced decrease in transepithelial resistance and restored claudin-8 protein level. In conclusion, barrier dysfunction in Y. enterocolitica infection is due to circumscribed epithelial TJ protein changes and necrotic cell loss, as a consequence of which leak flux diarrhea and antigen-uptake provoking extraintestinal arthritis may be triggered.

The Punicalagin Metabolites Ellagic Acid and Urolithin A Exert Different Strengthening and Anti-Inflammatory Effects on Tight Junction-Mediated Intestinal Barrier Function In Vitro.

Scope: Ellagitannins are polyphenols found in numerous fruits, nuts and seeds. The elagitannin punicalagin and its bioactive metabolites ellagic acid and urolithins are discussed to comprise a high potential for therapeutically or preventive medical application such as in intestinal diseases. The present study characterizes effects of punicalagin, ellagic acid and urolithin A on intestinal barrier function in the absence or presence of the proinflammatory cytokine tumor necrosis factor-α (TNFα). Methods and Results: Transepithelial resistance (TER), fluorescein and ion permeability, tight junction protein expression and signalling pathways were examined in Caco-2 and HT-29/B6 intestinal epithelial cell models. Punicalagin had less or no effects on barrier function in both cell models. Ellagic acid was most effective in ileum-like Caco-2 cells, where it increased TER and reduced fluorescein and sodium permeabilities. This was paralleled by myosin light chain kinase two mediated expression down-regulation of claudin-4, -7 and -15. Urolithin A impeded the TNFα-induced barrier loss by inhibition of claudin-1 and -2 protein expression upregulation and claudin-1 delocalization in HT-29/B6. Conclusion: Ellagic acid and urolithin A affect intestinal barrier function in distinct ways. Ellagic acid acts preventive by strengthening the barrier per se, while urolithin A protects against inflammation-induced barrier dysfunction.

A colonic mineralocorticoid receptor cell model expressing epithelial Na+ channels.

In the distal colon, the epithelial sodium channel (ENaC) is rate limiting for sodium absorption. Progress in the molecular characterization of ENaC expression and trafficking in response to the mineralocorticoid aldosterone has been hampered, since no epithelial colonic cell line existed expressing functional ENaC stimulated by nanomolar aldosterone via mineralocorticoid receptor (MR). Here, we present a human colonic epithelial cell line inducibly expressing the MR (HT-29/B6-Tet-On-MR) which exhibits aldosterone-dependent ENaC-mediated sodium transport in the presence of the short-chain fatty acid butyrate. Butyrate was necessary for high-level expression of MR which allowed for aldosterone-dependent upregulation of beta- and gamma-ENaC expression. As butyrate alone was not capable of promoting ENaC-mediated sodium transport, aldosterone-induced GILZ (glucocorticoid-induced leucine zipper protein) was identified as a candidate factor increasing apical ENaC levels.

Inflamed pouch mucosa possesses altered tight junctions indicating recurrence of inflammatory bowel disease.

BACKGROUND AND AIMS: The etiology of pouchitis after coloproctomucosectomy with ileal pouch-anal anastomosis in patients with ulcerative colitis is still unknown. Beside changes in luminal antigens, the immunological predisposition is assumed to be responsible. In previous electrophysiological studies, we showed that mucosal barrier and transport function in pouchitis is markedly reduced. Thus, the aim of the present study was to analyze barrier function on the molecular level. MATERIAL AND METHODS: Pouch biopsies of 36 ulcerative colitis patients were analyzed. Time points were (1) intraoperative immediately prior to ileal pouch-anal anastomosis (n = 13), (2) >1 year after ileostomy closure (pouch, n = 12), and (3) during pouchitis (n = 11). Control terminal ileum biopsies were obtained from eight patients undergoing hemicolectomy due to carcinoma. Expression of tight junction proteins was analyzed by Western blotting and confocal laser-scanning microscopy. To elucidate effects on epithelial barrier properties, impedance spectroscopy was performed in miniaturized Ussing chambers. RESULTS: In pouchitis, epithelial resistance was markedly reduced compared to non-inflamed pouch and control ileum. Expression of tight junction proteins claudin-1, 3, 4, 5, and 7 and occludin revealed differential expression regulation with the tightening tight junction protein claudin-1 being decreased and an increase of the pore-forming claudin-2, whereas other claudins remained constant. Morphometry indicated the mucosal surface to be unchanged. CONCLUSION: Pouchitis is characterized by a selective change of tight junction proteins in favor of opening the epithelial tight junction and, thus, the paracellular pathway, which contributes to the inflammatory process. This resembles changes in inflammatory bowel disease (IBD) and indicates IBD recurrence in pouchitis.

Phospholipid effects on SGLT1-mediated glucose transport in rabbit ileum brush border membrane vesicles.

In small intestine, sodium-glucose cotransporter SGLT1 provides the main mechanism for sugar uptake. We investigated the effect of membrane phospholipids (PL) on this transport in rabbit ileal brush border membrane vesicles (BBMV). For this, PL of different charge, length, and saturation were incorporated into BBMV. Transport was measured related to (i) membrane surface charge (membrane-bound MC540 fluorescence), (ii) membrane thickness (PL incorporation of different acyl chain length), and (iii) membrane fluidity (r12AS, fluorescence anisotropy of 12-AS). Compared to phosphatidylcholine (PC) carrying a neutral head group, inhibition of SGLT1 increased considerably with the acidic phosphatidic acid (PA) and phosphatidylinositol (PI) that increase membrane negative surface charge. The order of PL potency was PI>PA > PE = PS > PC. Inhibition by acidic PA-oleate was 5-times more effective than with neutral PE (phosphatidylethanolamine)-oleate. Lineweaver-Burk plot indicated uncompetitive inhibition of SGLT1 by PA. When membrane thickness was increased by neutral PC of varying acyl chain length, transport was increasingly inhibited by 16:1 PC to 22:1 PC. Even more pronounced inhibition was observed with mono-unsaturated instead of saturated acyl chains which increased membrane fluidity (indicated by decreased r12AS). In conclusion, sodium-dependent glucose transport of rabbit ileal BBMV is modulated by (i) altered membrane surface charge, (ii) length of acyl chains via membrane thickness, and (iii) saturation of PL acyl chains altering membrane fluidity. Transport was attenuated by charged PL with longer and unsaturated acyl residues. Alterations of PL may provide a principle for attenuating dietary glucose uptake.

Campylobacter jejuni enters gut epithelial cells and impairs intestinal barrier function through cleavage of occludin by serine protease HtrA.

Campylobacter jejuni secretes HtrA (high temperature requirement protein A), a serine protease that is involved in virulence. Here, we investigated the interaction of HtrA with the host protein occludin, a tight junction strand component. Immunofluorescence studies demonstrated that infection of polarized intestinal Caco-2 cells with C. jejuni strain 81-176 resulted in a redistribution of occludin away from the tight junctions into the cytoplasm, an effect that was also observed in human biopsies during acute campylobacteriosis. Occludin knockout Caco-2 cells were generated by CRISPR/Cas9 technology. Inactivation of this gene affected the polarization of the cells in monolayers and transepithelial electrical resistance (TER) was reduced, compared to wild-type Caco-2 cells. Although tight junctions were still being formed, occludin deficiency resulted in a slight decrease of the tight junction plaque protein ZO-1, which was redistributed off the tight junction into the lateral plasma membrane. Adherence of C. jejuni to Caco-2 cell monolayers was similar between the occludin knockout compared to wild-type cells, but invasion was enhanced, indicating that deletion of occludin allowed larger numbers of bacteria to pass the tight junctions and to reach basal membranes to target the fibronectin receptor followed by cell entry. Finally, we discovered that purified C. jejuni HtrA cleaves recombinant occludin in vitro to release a 37 kDa carboxy-terminal fragment. The same cleavage fragment was observed in Western blots upon infection of polarized Caco-2 cells with wild-type C. jejuni, but not with isogenic ΔhtrA mutants. HtrA cleavage was mapped to the second extracellular loop of occludin, and a putative cleavage site was identified. In conclusion, HtrA functions as a secreted protease targeting the tight junctions, which enables the bacteria by cleaving occludin and subcellular redistribution of other tight junction proteins to transmigrate using a paracellular mechanism and subsequently invade epithelial cells.

Quercetin enhances epithelial barrier function and increases claudin-4 expression in Caco-2 cells.

Quercetin is the most abundant flavonoid and is assumed to have positive effects on the gastrointestinal mucosa after dietary intake. The aim of the study was to analyze the influence of quercetin on intestinal barrier function using the human colonic epithelial cell line Caco-2. Transepithelial resistance (R(t)), tracer fluxes of [(3)H]-mannitol, 22Na+, and 36Cl- as well as electrogenic ion transport were determined in Ussing chambers. Expression of tight junction (TJ) proteins and mRNA was analyzed in Western blots and quantitative RT-PCR, respectively. Regulation of transcription was analyzed by reporter gene assay. Cellular distribution of TJ proteins was examined by confocal laser scanning microscopy (LSM). Apoptotic rate was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Quercetin induced a dose-dependent increase of R(t) persisting for >2 d. Daily addition of quercetin was able to perpetuate the effect, which was seen whether quercetin was added apically or to the basolateral compartment. Parallel to the R(t) increase, quercetin induced a strong increase of the TJ protein claudin-4 but not of other claudins. Confocal LSM showed a localization of claudin-4 in TJ. Apoptotic rate was not affected by quercetin. Consistent with these changes, fluxes of Na+ and Cl-, but not of mannitol, were reduced. Reporter gene assays revealed a stimulatory effect of quercetin on claudin-4 transcription. The flavonoid quercetin enhances barrier function via transcriptional expression regulation of the TJ protein claudin-4, which represents an important protective effect of this food component against barrier disturbance in intestinal inflammation.

Reversible opening of the blood-brain barrier by claudin-5-binding variants of Clostridium perfringens enterotoxin's claudin-binding domain.

The blood-brain barrier (BBB) prevents entry of neurotoxic substances but also that of drugs into the brain. Here, the paracellular barrier is formed by tight junctions (TJs) with claudin-5 (Cldn5) being the main sealing constituent. Transient BBB opening by targeting Cldn5 could improve paracellular drug delivery. The non-toxic C-terminal domain of Clostridium perfringens enterotoxin (cCPE) binds to a subset of claudins, e.g., Cldn3, -4. Structure-based mutagenesis was used to generate Cldn5-binding variants (cCPE-Y306W/S313H and cCPE-N218Q/Y306W/S313H). These cCPE-variants were tested for transient TJ opening using multiple in vitro BBB models: Primary porcine brain endothelial cells, coculture of primary rat brain endothelial cells with astrocytes and mouse cerebEND cells. cCPE-Y306W/S313H and cCPE-N218Q/Y306W/S313H but neither cCPE-wt nor cCPE-Y306A/L315A (not binding to claudins) decreased transendothelial electrical resistance in a concentration-dependent and reversible manner. Furthermore, permeability of carboxyfluorescein (with size of CNS drugs) was increased. cCPE-Y306W/S313H but neither cCPE-wt nor cCPE-Y306A/L315A bound to Cldn5-expressing brain endothelial cells. However, freeze-fracture EM showed that cCPE-Y306W/S313H did not cause drastic TJ breakdown. In sum, Cldn5-binding cCPE-variants enabled mild and transient opening of brain endothelial TJs. Using reliable in vitro BBB models, the results demonstrate that cCPE-based biologics designed to bind Cldn5 improve paracellular drug delivery across the BBB.

Celiac Disease: Role of the Epithelial Barrier.

In celiac disease (CD) a T-cell-mediated response to gluten is mounted in genetically predisposed individuals, resulting in a malabsorptive enteropathy histologically highlighted by villous atrophy and crypt hyperplasia. Recent data point to the epithelial layer as an under-rated hot spot in celiac pathophysiology to date. This overview summarizes current functional and genetic evidence on the role of the epithelial barrier in CD, consisting of the cell membranes and the apical junctional complex comprising sealing as well as ion and water channel-forming tight junction proteins and the adherens junction. Moreover, the underlying mechanisms are discussed, including apoptosis of intestinal epithelial cells, biology of intestinal stem cells, alterations in the apical junctional complex, transcytotic uptake of gluten peptides, and possible implications of a defective epithelial polarity. Current research is directed toward new treatment options for CD that are alternatives or complementary therapeutics to a gluten-free diet. Thus, strategies to target an altered epithelial barrier therapeutically also are discussed.

Lactoferrin protects against intestinal inflammation and bacteria-induced barrier dysfunction in vitro.

The iron-binding glycoprotein lactoferrin (LF) is naturally present in human breast milk. Several studies suggest that LF contributes to infant health and development owing to a variety of protective effects, including antimicrobial and anti-inflammatory features. Therefore, we aimed to elucidate its protective properties on intestinal epithelial barrier dysfunction induced by infection or inflammation using the human epithelial cell culture models HT-29/B6 and T84. During barrier perturbation induced by the proinflammatory cytokine tumor necrosis factor α (TNF-α), bovine LF restored tight junction (TJ) morphometry and inhibited TNF-α-induced epithelial apoptosis. This resulted in an attenuation of the TNF-α-induced decrease in transepithelial resistance (TER) and increases in permeability of fluorescein and FITC-dextran (4 kDa) and was as effective as the apoptosis inhibitor Q-VD-Oph. The enteropathogenic bacterium Yersinia enterocolitica is a frequent cause of diarrhea in early childhood. This involves focal changes in TJ protein expression and localization. LF diminished the Y. enterocolitica-induced drop in TER in the present in vitro model, which was paralleled by an inhibition of the Yersinia-induced reduction of claudin-8 expression via c-Jun kinase signaling. In conclusion, LF exerts protective effects against inflammation- or infection-induced barrier dysfunction in human intestinal cell lines, supporting its relevance for healthy infant development.