Vasoactive intestinal peptide induces IL-8 production in human colonic epithelial cells via MAP kinase-dependent and PKA-independent pathways

Vasoactive intestinal peptide (VIP) has been shown to be a key regulator of intestinal epithelial functions such as mucus and chloride secretion, paracellular permeability, and cell proliferation. However, its regulatory role in intestinal epithelial chemokine production remains unknown. The aim of this study was (1) to determine whether VIP can modulate intestinal epithelial interleukin-8 (IL-8) production and (2) to identify intracellular mediators responsible for this effect. In the human colonic epithelial cell line HT29-Cl.16E, VIP stimulates IL-8 secretion dose-dependently and IL-8 mRNA level at 10(-9) M. The protein kinase A (PKA) inhibitor PKI did not abolish the effect of VIP. However, inhibition of the ERK1/2 and p38 MAPK pathways reduced the VIP-stimulated IL-8 secretion and mRNA level. Together, our results showed that VIP stimulates IL-8 production in intestinal epithelial cells via PKA-independent and MAPK-dependent pathways. These data suggest that VIPergic pathways can play an immunomodulatory role in intestinal epithelial cells, by regulating epithelial IL-8 secretion.     

TFF3-peptide increases transepithelial resistance in epithelial cells by modulating claudin-1 and -2 expression

TFF3 plays an important role in the protection and repair of the gastrointestinal mucosa. The molecular mechanisms of TFF function, however, are still largely unknown. Increasing evidence indicates that apart from stabilizing mucosal mucins TFF3 induces cellular signals that modulate cell–cell junctions of epithelia. In transfected HT29/B6 and MDCK cells stably expressing FLAG-tagged human TFF3 we have recently shown that TFF3 down-regulates E-cadherin, impairs the function of adherens junctions and thus facilitates cell migration in wounded epithelial cell layers. Here we investigate TFF3-induced effects on the composition and function of tight junctions in these cells. TFF3 increased the cellular level of tightening claudin-1 and decreased the amount of claudin-2 known to form cation-selective channels. Expression of ZO-1, ZO-2 and occludin was not altered. The change in claudin-1 and -2 expression in TFF3-expressing HT29/B6 cells was accompanied by an increase in the transepithelial resistance in confluent monolayers of these cells. These data suggest that TFF3 plays a role in the regulation of intestinal barrier function by altering the claudin composition within tight junctions thus decreasing paracellular permeability of the intestinal mucosa.     

Differential role of Rho GTPases in intestinal epithelial barrier regulation in vitro

Maintenance of intestinal epithelial barrier functions is crucial to prevent systemic contamination by microbes that penetrate from the gut lumen. GTPases of the Rho-family such as RhoA, Rac1, and Cdc42 are known to be critically involved in the regulation of intestinal epithelial barrier functions. However, it is still unclear whether inactivation or activation of these GTPases exerts barrier protection or not. We tested the effects of Rho GTPase activities on intestinal epithelial barrier functions by using the bacterial toxins cytotoxic necrotizing factor 1 (CNF-1), toxin B, C3 transferase (C3 TF), and lethal toxin (LT) in an in vitro model of the intestinal epithelial barrier. Incubation of cell monolayers with CNF-1 for 3 h induced exclusive activation of RhoA whereas Rac1 and Cdc42 activities were unchanged. As revealed by FITC-dextran flux and measurements of transepithelial electrical resistance (TER) intestinal epithelial permeability was significantly increased under these conditions. Inhibition of Rho kinase via Y27632 blocked barrier destabilization of CNF-1 after 3 h. In contrast, after 24 h of incubation with CNF-1 only Rac1 and Cdc42 but not RhoA were activated which resulted in intestinal epithelial barrier stabilization. Toxin B to inactivate RhoA, Rac1, and Cdc42 as well as Rac1 inhibitor LT increased intestinal epithelial permeability. Similar effects were observed after inhibition of RhoA/Rho kinase signaling by C3 TF or Y27632. Taken together, these data demonstrate that both activation and inactivation of RhoA signaling increased paracellular permeability whereas activation of Rac1 and Cdc42 correlated with stabilized barrier functions.     

Increased iNOS activity is essential for pulmonary epithelial tight junction dysfunction in endotoxemic mice

A murine endotoxemia model and cultured Calu-3 monolayers were used to test the hypothesis that excessive nitric oxide (NO) production secondary to induction of inducible NO synthase (iNOS) is a key factor leading to altered tight junction (TJ) protein expression and function in the pulmonary epithelium. C57Bl/6J mice were injected with either Escherichia coli 0111:B4 lipopolysaccharide (LPS; 2 mg/kg) or vehicle. Twelve hours later, leakage of FITC-dextran (M(r) 4 kDa; FD4) from blood into bronchoalveolar lavage fluid was significantly increased in endotoxemic but not control mice. This decrease in bronchoalveolar barrier function was associated with upregulation of iNOS protein expression and NF-kappaB activation in lung tissue. Expression of the TJ proteins, zonula occludens (ZO)-1, ZO-2, ZO-3, and occludin, as assessed by immunoblotting and/or immunofluorescence, decreased in lung after the injection of mice with LPS. Treatment of endotoxemic mice with an isoform-selective iNOS inhibitor [l-N(6)-(1-iminoethyl)lysine; l-NIL] ameliorated LPS-induced changes in TJ protein expression and preserved bronchoalveolar epithelial barrier function. Incubating Calu-3 bronchiolar epithelial monolayers with cytomix (a mixture of 1,000 U/ml IFN-gamma, 10 ng/ml TNF-alpha, and 1 ng/ml IL-1beta) increased permeability to FD4, but adding l-NIL prevented this effect. These results suggest that decreased expression and mistargeting of TJ proteins in lung after systemic inflammation may be NO dependent.     

Lipopolysaccharide disrupts tight junctions in cholangiocyte monolayers by a c-Src-, TLR4-, and LBP-dependent mechanism

Bile duct epithelium forms a barrier to the backflow of bile into the liver parenchyma. However, the structure and regulation of the tight junctions in bile duct epithelium is not well understood. In the present study, we evaluated the effect of lipopolysaccharide on tight junction integrity and barrier function in normal rat cholangiocyte monolayers. Lipopolysaccharide disrupts barrier function and increases paracellular permeability in a time- and dose-dependent manner. Lipopolysaccharide induced a redistribution of tight junction proteins, occludin, claudin-1, claudin-4, and zonula occludens (ZO)-1 from the intercellular junctions and reduced the level of ZO-1. Tyrosine kinase inhibitors (genistein and PP2) prevented lipopolysaccharide-induced increase in permeability and subcellular redistribution of ZO-1. Reduced expression of c-Src, TLR4, or LBP by specific small interfering RNA attenuated lipopolysaccharide-induced permeability and redistribution of ZO-1. ML-7, a myosin light chain kinase inhibitor, attenuated LPS-induced permeability. Lipopolysaccharide treatment rapidly increased the phosphorylation of occludin and ZO-1 on tyrosine residues, which was prevented by genistein and PP2. Occludin and ZO-1 were found to be highly phosphorylated on threonine residues in intact cell monolayers. Threonine-phosphorylation of occludin was rapidly reduced by lipopolysaccharide administration. Lipopolysaccharide-induced dephosphorylation of occludin on Thr residues was prevented by genistein and PP2. In conclusion, lipopolysaccharide disrupts the tight junction of a bile duct epithelial monolayer by a c-Src-, TLR4-, LBP-, and myosin light chain kinase-dependent mechanism.     

The role of caspase-3 in lipopolysaccharide-mediated disruption of intestinal epithelial tight junctions

The mechanisms responsible for microbially induced epithelial apoptosis and increased intestinal permeability remain unclear. This study assessed whether purified bacterial lipopolysaccharide (LPS) increases epithelial apoptosis and permeability and whether these changes are dependent on caspase-3 activation. In nontumorigenic epithelial monolayers, Escherichia coli O26:B6 LPS increased apoptosis, as shown by nuclear breakdown, caspase-3 activation, and PARP cleavage, and induced disruption of tight junctional ZO-1. Apical, but not basolateral, exposure to LPS increased epithelial permeability. Addition of a caspase-3 inhibitor abolished the effects of LPS. The findings describe a novel mechanism whereby apical LPS may disrupt epithelial tight junctional ZO-1 and barrier function in a caspase-3-dependent fashion.     

Neuroplasticity and neuroprotection in enteric neurons: Role of epithelial cells

Neurons of enteric nervous system (ENS) regulate intestinal epithelial cells (IEC) functions but whether IEC can impact upon the neurochemical coding and survival of enteric neurons remain unknown. Neuro-epithelial interactions were studied using a coculture model composed of IEC lines and primary culture of rat ENS or human neuroblastoma cells (SH-SY5Y). Neurochemical coding of enteric neurons was analysed by immunohistochemistry and quantitative PCR. Neuroprotective effects of IEC were tested by measuring neuron specific enolase (NSE) release or cell permeability to 7-amino-actinomycin D (7-AAD). Following coculture with IEC, the percentage of VIP-immunoreactive (IR) neurons but not NOS-IR and VIP mRNA expression were significantly increased. IEC significantly reduced dopamine-induced NSE release and 7-AAD permeability in culture of ENS and SH-SY5Y, respectively. Finally, we showed that NGF had neuroprotective effects but reduced VIP expression in enteric neurons. In conclusion, our study identified a novel role for IEC in the regulation of enteric neuronal properties.     

Expression and distribution of symplekin regulates the assembly and function of the epithelial tight junction

Symplekin is multifunctional protein localized to both the tight junction and the nucleus with known roles in mRNA polyadenylation, proliferation, differentiation and tumorigenesis. Functions of symplekin at tight junctions have not been systematically investigated. In this study, increased expression of symplekin was observed during the formation of tight junctions in cultured HT-29 and HepG2 epithelial cells. Repression of symplekin by RNAi increased the permeability of epithelial monolayers, disrupted cellular polarity, and decreased the expression of the tight junction protein ZO-1. Moreover, symplekin was co-localized with ZO-1 at tight junctions and co-immunoprecipitated with ZO-1, indicating that ZO-1 and symplekin form complexes. In conclusion, symplekin expression regulates the assembly of tight junctions, thus helps to maintain the integrity of the epithelial monolayer and cellular polarity.     

Salmonella enterica serovar Typhimurium effectors SopB, SopE, SopE2 and SipA disrupt tight junction structure and function

Salmonella enterica serovar Typhimurium is a major cause of human gastroenteritis. Infection of epithelial monolayers by S. Typhimurium disrupts tight junctions that normally maintain the intestinal barrier and regulate cell polarity. Tight junction disruption is dependent upon the Salmonella pathogenicity island-1 (SPI-1) type 3 secretion system but the specific effectors involved have not been identified. In this study we demonstrate that SopB, SopE, SopE2 and SipA are the SPI-1-secreted effectors responsible for disruption of tight junction structure and function. Tight junction disruption by S. Typhimurium was prevented by inhibiting host protein geranylgeranylation but was not dependent on host protein synthesis or secretion of host-derived products. Unlike wild-type S. Typhimurium, DeltasopB, DeltasopE/E2, DeltasipA, or DeltasipA/sopB mutants, DeltasopB/E/E2 and DeltasipA/sopE/E2 mutants were unable to increase the permeability of polarized epithelial monolayers, did not disrupt the distribution or levels of ZO-1 and occludin, and did not alter cell polarity. These data suggest that SPI-1-secreted effectors utilize their ability to stimulate Rho family GTPases to disrupt tight junction structure and function.     

Enteric Glia Cells Attenuate Cytomix-Induced Intestinal Epithelial Barrier Breakdown

Background

Intestinal barrier failure may lead to systemic inflammation and distant organ injury in patients following severe injury. Enteric glia cells (EGCs) have been shown to play an important role in maintaining gut barrier integrity through secretion of S-Nitrosoglutathione (GSNO). We have recently shown than Vagal Nerve Stimulation (VNS) increases EGC activation, which was associated with improved gut barrier integrity. Thus, we sought to further study the mechanism by which EGCs prevent intestinal barrier breakdown utilizing an in vitro model. We postulated that EGCs, through the secretion of GSNO, would improve intestinal barrier function through improved expression and localization of intestinal tight junction proteins.

Methods

Epithelial cells were co-cultured with EGCs or incubated with GSNO and exposed to Cytomix (TNF-α, INF-γ, IL-1β) for 24 hours. Barrier function was assessed by permeability to 4kDa FITC-Dextran. Changes in tight junction proteins ZO-1, occludin, and phospho-MLC (P-MLC) were assessed by immunohistochemistry and immunoblot.

Key Results

Co-culture of Cytomix-stimulated epithelial monolayers with EGCs prevented increases in permeability and improved expression and localization of occludin, ZO-1, and P-MLC. Further, treatment of epithelial monolayers with GSNO also prevented Cytomix-induced increases in permeability and exhibited a similar improvement in expression and localization of occludin, ZO-1, and P-MLC.

Conclusions & Inferences

The addition of EGCs, or their secreted mediator GSNO, prevents epithelial barrier failure after injury and improved expression of tight junction proteins. Thus, therapies that increase EGC activation, such as VNS, may be a novel strategy to limit barrier failure in patients following severe injury.     

TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation

Crohn's disease (CD) patients have an abnormal increase in intestinal epithelial permeability. The defect in intestinal tight junction (TJ) barrier has been proposed as an important etiologic factor of CD. TNF-alpha increases intestinal TJ permeability. Because TNF-alpha levels are markedly increased in CD, TNF-alpha increase in intestinal TJ permeability could be a contributing factor of intestinal permeability defect in CD. Our purpose was to determine some of the intracellular mechanisms involved in TNF-alpha modulation of intestinal epithelial TJ permeability by using an in vitro intestinal epithelial system consisting of filter-grown Caco-2 monolayers. TNF-alpha produced a concentration- and time-dependent increase in Caco-2 TJ permeability. TNF-alpha-induced increase in Caco-2 TJ permeability correlated with Caco-2 NF-kappa B activation. Inhibition of TNF-alpha-induced NF-kappa B activation by selected NF-kappa B inhibitors, curcumin and triptolide, prevented the increase in Caco-2 TJ permeability, indicating that NF-kappa B activation was required for the TNF-alpha-induced increase in Caco-2 TJ permeability. This increase in Caco-2 TJ permeability was accompanied by down-regulation of zonula occludens (ZO)-1 proteins and alteration in junctional localization of ZO-1 proteins. TNF-alpha modulation of ZO-1 protein expression and junctional localization were also prevented by NF-kappa B inhibitors. TNF-alpha did not induce apoptosis in Caco-2 cells, suggesting that apoptosis was not the mechanism involved in TNF-alpha-induced increase in Caco-2 TJ permeability. These results demonstrate for the first time that TNF-alpha-induced increase in Caco-2 TJ permeability was mediated by NF-kappa B activation. The increase in permeability was associated with NF-kappa B-dependent downregulation of ZO-1 protein expression and alteration in junctional localization.     

Statin pleiotropy prevents rho kinase-mediated intestinal epithelial barrier compromise induced by Blastocystis cysteine proteases

Blastocystis is an enteric parasite that causes acute and chronic intestinal infections, often non-responsive to conventional antibiotics. The effects of Blastocystis infections on human epithelial permeability are not known, and molecular mechanisms of Blastocystis-induced intestinal pathology remain unclear. This study was conducted to determine whether Blastocystis species alters human intestinal epithelial permeability, to assess whether these abnormalities are rho kinase (ROCK)-dependent, and to investigate the therapeutic potential of the HMG-CoA reductase inhibitor Simvastatin in altered intestinal epithelial barrier function. The effect of metronidazole resistant (Mz(r) ) Blastocystis isolated from a symptomatic patient on human colonic epithelial monolayers (Caco-2) was assessed. Modulation of enterocyte myosin light chain phosphorylation, transepithelial fluorescein isothiocyanate-dextran fluxes, transepithelial resistance, cytoskeletal F-actin and tight junctional zonula occludens-1 (ZO-1) by parasite cysteine proteases were measured in the presence or absence of HMG-CoA reductase and ROCK inhibition. Blastocystis significantly decreased transepithelial resistance, increased epithelial permeability, phosphorylated myosin light chain and reorganized epithelial actin cytoskeleton andZO-1. Thesealterations were abolished byinhibition of enterocyte ROCK, HMG-CoA reductase and parasite cysteine protease. Our findings suggest that cysteine proteases of Mz(r) Blastocystis induce ROCK-dependent disruption of intestinal epithelial barrier function and correlates with reorganization of cytoskeletal F-actin and tight junctional ZO-1. Simvastatin prevented parasite-induced barriercompromise, suggesting a therapeutic potential of statins in intestinal infections.     

Nitric oxide attenuates hydrogen peroxide-induced barrier disruption and protein tyrosine phosphorylation in monolayers of intestinal epithelial cell

The intestinal epithelium provides a barrier to the transport of harmful luminal molecules into the systemic circulation. A dysfunctional epithelial barrier is closely associated with the pathogenesis of a variety of intestinal and systemic disorders. We investigated here the effects of nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) on the barrier function of a human intestinal epithelial cell line, Caco-2. When treated with H(2)O(2), Caco-2 cell monolayers grown on permeable supports exhibited several remarkable features of barrier dysfunction as follows: a decrease in transepithelial electrical resistance, an increase in paracellular permeability to dextran, and a disruption of the intercellular junctional localization of the scaffolding protein ZO-1. In addition, an induction of tyrosine phosphorylation of numerous cellular proteins including ZO-1, E-cadherin, and beta-catenin, components of tight and adherens junctions, was observed. On the other hand, combined treatment of Caco-2 monolayers with H(2)O(2) and an NO donor (NOC5 or NOC12) relieved the damage to the barrier function and suppressed the protein tyrosine phosphorylation induced by H(2)O(2) alone. These results suggest that NO protects the barrier function of intestinal epithelia from oxidative stress by modulating some intracellular signaling pathways of protein tyrosine phosphorylation in epithelial cells.     

Glutamine regulates Caco-2 cell tight junction proteins

Intestinal epithelial tight junction (TJ) barrier dysfunction may lead to inflammation and mucosal injury. Glutamine (GLN) plays a role in maintenance of intestinal barrier function in various animal models and critically ill humans. Recent evidence from intestinal cell monolayers indicates that GLN maintains transepithelial resistance and decreases permeability. The mechanisms of these effects remain undefined. We hypothesized that GLN affects proteins involved in the intercellular junctional complex. GLN availability was controlled in Caco-2 monolayers by addition to the medium and treatment with methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Expression of TJ proteins, claudin-1, occludin, and zonula occluden (ZO)-1 was measured by immunoblotting. Localization of TJ proteins was evaluated by immunofluorescence light microscopy. Structure of TJ was determined by transmission electron microscopy (TEM). Deprivation of GLN decreased claudin-1, occludin, and ZO-1 protein expression and caused a disappearance of perijunctional claudin-1 and a reduction of occludin but had no effect on ZO-1. TEM revealed that MSO-treated cells in the absence of GLN formed irregular junctional complexes between the apical lateral margins of adjoining cells. These findings indicate that TJ protein expression and cellular localization in Caco-2 cell monolayers rely on GLN. This mechanism may similarly relate to GLN-mediated modulation of intestinal barrier function in stressed animals and humans.     

Occludin deficiency promotes ethanol-induced disruption of colonic epithelial junctions,gut barrier dysfunction and liver damage in mice

BackgroundDisruption of epithelial tight junctions (TJ), gut barrier dysfunction and endotoxemia play crucial role in the pathogenesis of alcoholic tissue injury. Occludin, a transmembrane protein of TJ, is depleted in colon by alcohol. However, it is unknown whether occludin depletion influences alcoholic gut and liver injury.MethodsWild type (WT) and occludin deficient (Ocln^−/−) mice were fed 1–6% ethanol in Lieber–DeCarli diet. Gut permeability was measured by vascular-to-luminal flux of FITC-inulin. Junctional integrity was analyzed by confocal microscopy. Liver injury was assessed by plasma transaminase, histopathology and triglyceride analyses. The effect of occludin depletion on acetaldehyde-induced TJ disruption was confirmed in Caco-2 cell monolayers.ResultsEthanol feeding significantly reduced body weight gain in Ocln^−/− mice. Ethanol increased inulin permeability in colon of both WT and Ocln^−/− mice, but the effect was 4-fold higher in Ocln^−/− mice. The gross morphology of colonic mucosa was unaltered, but ethanol disrupted the actin cytoskeleton, induced redistribution of occludin, ZO-1, E-cadherin and β-catenin from the junctions and elevated TLR4, which was more severe in Ocln^−/− mice. Occludin knockdown significantly enhanced acetaldehyde-induced TJ disruption and barrier dysfunction in Caco-2 cell monolayers. Ethanol significantly increased liver weight and plasma transaminase activity in Ocln^−/− mice, but not in WT mice. Histological analysis indicated more severe lesions and fat deposition in the liver of ethanol-fed Ocln^−/− mice. Ethanol-induced elevation of liver triglyceride was also higher in Ocln^−/− mice.ConclusionThis study indicates that occludin deficiency increases susceptibility to ethanol-induced colonic mucosal barrier dysfunction and liver damage in mice.     

Protein Kinase D Promotes Airway Epithelial Barrier Dysfunction and Permeability through Down-regulation of Claudin-1

At the interface between host and external environment, the airway epithelium serves as a major protective barrier. In the present study we show that protein kinase D (PKD) plays an important role in the formation and integrity of the airway epithelial barrier. Either inhibition of PKD activity or silencing of PKD increased transepithelial electrical resistance (TEER), resulting in a tighter epithelial barrier. Among the three PKD isoforms, PKD3 knockdown was the most efficient one to increase TEER in polarized airway epithelial monolayers. In contrast, overexpression of PKD3 wild type, but not PKD3 kinase-inactive mutant, disrupted the formation of apical intercellular junctions and their reassembly, impaired the development of TEER, and increased paracellular permeability to sodium fluorescein in airway epithelial monolayers. We further found that overexpression of PKD, in particular PKD3, markedly suppressed the mRNA and protein levels of claudin-1 but had only minor effects on the expression of other tight junctional proteins (claudin-3, claudin-4, claudin-5, occludin, and ZO-1) and adherent junctional proteins (E-cadherin and β-catenin). Immunofluorescence study revealed that claudin-1 level was markedly reduced and almost disappeared from intercellular contacts in PKD3-overexpressed epithelial monolayers and that claudin-4 was also restricted from intercellular contacts and tended to accumulate in the cell cytosolic compartments. Last, we found that claudin-1 knockdown prevented TEER elevation by PKD inhibition or silencing in airway epithelial monolayers. These novel findings indicate that PKD negatively regulates human airway epithelial barrier formation and integrity through down-regulation of claudin-1, which is a key component of tight junctions.     

Protective Effects of Carbon Monoxide-Releasing Molecule-2 on the Barrier Function of Intestinal Epithelial Cells

Objective

To investigate the protective effects and mechanisms of carbon monoxide-releasing molecule-2 (CORM-2) on barrier function of intestinal epithelial cells.

Materials and Methods

After pre-incubation with CORM-2 for 1 hour, cultured intestinal epithelial IEC-6 cells were stimulated with 50 µg/ml lipopolysaccharides (LPS). Cytokines levels in culture medium were detected using ELISA kits. Trans-epithelial electrical resistance (TER) of IEC-6 cell monolayers in Transwells were measured with a Millipore electric resistance system (ERS-2; Millipore) and calculated as Ω/cm² at different time points after LPS treatment. The permeability changes were also measured using FITC-dextran. The levels of tight junction (TJ) proteins (occludin and ZO-1) and myosin light chain (MLC) phosphorylation were detected using Western blotting with specific antibodies. The subsequent structural changes of TJ were visualized using transmission electron microscopy (TEM).

Results

CORM-2 significantly reduced LPS-induced secretion of TNF-α and IL-1β. The LPS-induced decrease of TER and increase of permeability to FITC-dextran were inhibited by CORM-2 in a concentration dependent manner (P<0.05). LPS-induced reduction of tight junction proteins and increase of MLC phosphorylation were also attenuated. In LPS-treated cells, TEM showed diminished electron-dense material and interruption of TJ and desmosomes between the apical lateral margins of adjoining cells, which were prevented by CORM-2 treatment.

Conclusions

The present study demonstrates that CORM-2, as a novel CO-releasing molecule, has ability to protect the barrier function of LPS-stimulated intestinal epithelial cells. Inhibition of inflammatory cytokines release, restoration of TJ proteins and suppression of MLC phosphorylation are among the protective effects of CORM-2.     

Oral and fecal Campylobacter concisus strains perturb barrier function by apoptosis induction in HT-29/B6 intestinal epithelial cells

Campylobacter concisus infections of the gastrointestinal tract can be accompanied by diarrhea and inflammation, whereas colonization of the human oral cavity might have a commensal nature. We focus on the pathophysiology of C. concisus and the effects of different clinical oral and fecal C. concisus strains on human HT-29/B6 colon cells. Six oral and eight fecal strains of C. concisus were isolated. Mucus-producing HT-29/B6 epithelial monolayers were infected with the C. concisus strains. Transepithelial electrical resistance (R(t)) and tracer fluxes of different molecule size were measured in Ussing chambers. Tight junction (TJ) protein expression was determined by Western blotting, and subcellular TJ distribution was analyzed by confocal laser-scanning microscopy. Apoptosis induction was examined by TUNEL-staining and Western blot of caspase-3 activation. All strains invaded confluent HT-29/B6 cells and impaired epithelial barrier function, characterized by a time- and dose-dependent decrease in R(t) either after infection from the apical side but even more from the basolateral compartment. TJ protein expression changes were sparse, only in apoptotic areas of infected monolayers TJ proteins were redistributed. Solely the barrier-forming TJ protein claudin-5 showed a reduced expression level to 66±8% (P<0.05), by expression regulation from the gene. Concomitantly, Lactate dehydrogenase release was elevated to 3.1±0.3% versus 0.7±0.1% in control (P<0.001), suggesting cytotoxic effects. Furthermore, oral and fecal C. concisus strains elevated apoptotic events to 5-fold. C. concisus-infected monolayers revealed an increased permeability for 332 Da fluorescein (1.74±0.13 vs. 0.56±0.17 10(-6) cm/s in control, P<0.05) but showed no difference in permeability for 4 kDa FITC-dextran (FD-4). The same was true in camptothecin-exposed monolayers, where camptothecin was used for apoptosis induction.In conclusion, epithelial barrier dysfunction by oral and fecal C. concisus strains could mainly be assigned to apoptotic leaks together with moderate TJ changes, demonstrating a leak-flux mechanism that parallels the clinical manifestation of diarrhea.