Arcobacter butzleri isolates exhibit pathogenic potential in intestinal epithelial cell models.

AIMS: The pathogenic potential of Arcobacter butzleri isolates on human (HT-29/B6) and porcine epithelial (IPEC-J2) cells was investigated by in vitro assays. METHODS AND RESULTS: Five of six A. butzleri isolates were able to adhere and invade HT-29/B6 cells while only four isolates adhered and two invaded IPEC-J2 cells. Two non- or poorly invasive A. butzleri isolates were highly cytotoxic to differentiated HT-29/B6 cells but none to IPEC-J2 cells as determined by WST-assays. Epithelial integrity of cell monolayers, monitored by measurement of the transepithelial electrical resistance (TER), was decreased by all A. butzleri isolates in HT-29/B6 and IPEC-J2 cells to 30-15% and 90-50% respectively. CONCLUSION: The A. butzleri strain-specific pathomechanisms observed with the human colon cell line HT-29/B6, like adhesion, invasion and cytotoxicity might all contribute to epithelial barrier dysfunction, which could explain a leak-flux type of diarrhoea in humans. In contrast, porcine cells seem to be less susceptible to A. butzleri. SIGNIFICANCE AND IMPACT OF THE STUDY: Arcobacter butzleri has enteric pathogenic potential, characterized by defined interactions with human epithelial cells and strain-specific pathomechanisms.

Resveratrol Prevents Campylobacter jejuni-Induced Leaky gut by Restoring Occludin and Claudin-5 in the Paracellular Leak Pathway.

Campylobacter jejuni is a bacterial human pathogen causing gastroenteritis and sequelae like irritable bowel syndrome. Epidemiologists count the human campylobacteriosis by C. jejuni as the most common foodborne zoonosis and bacterial diarrheal disease worldwide. Based on bioinformatics predictions for potential protective compounds in campylobacteriosis, the question was raised whether the plant-based polyphenol resveratrol is sufficient to attenuate intestinal epithelial damage induced by C. jejuni. We investigated this by performing experimental infection studies in an epithelial cell culture and the secondary abiotic IL-10-/- mouse model. In C. jejuni-infected human colonic HT-29/B6 cell monolayers, transepithelial electrical resistance (TER) was decreased and the paracellular marker flux of fluorescein (332 Da) increased. Concomitantly, the tight junction (TJ) proteins occludin and claudin-5 were re-distributed off the tight junction domain. This was accompanied by an increased induction of epithelial apoptosis, both changes contributing to compromised barrier function and the opening of the leak pathway induced by C. jejuni. In parallel, the recovery experiments with the application of resveratrol revealed a functional improvement of the disturbed epithelial barrier in both models in vitro and in vivo. During treatment with resveratrol, TJ localization of occludin and claudin-5 was fully restored in the paracellular domain of HT-29/B6 cells. Moreover, resveratrol decreased the rate of epithelial apoptosis. These resveratrol-induced molecular and cellular effects would therefore be expected to improve epithelial barrier function, thereby minimizing the so-called leaky gut phenomenon. In conclusion, the induction of the leak pathway by C. jejuni and the restoration of barrier function by resveratrol demonstrates its effectiveness as a potential preventive or therapeutic method of mitigating the leaky gut associated with campylobacteriosis.

Structural and functional changes of the duodenum in human norovirus infection.

BACKGROUND: Norovirus infection is the most frequent cause of infectious diarrhoea in the western world. This study aimed to characterise functionally and histomorphologically the diseased duodenum in human biopsies. METHODS: Norovirus infection was diagnosed by the Kaplan criteria and confirmed by PCR of stool samples. Duodenal biopsies were obtained endoscopically. In miniaturised Ussing chambers, short circuit current, flux measurements and impedance spectroscopy were performed. Histological analysis including apoptosis staining and characterisation of intraepithelial lymphocytes was performed. Tight junction proteins were quantified by immunoblotting. RESULTS: In norovirus infection, epithelial resistance decreased from (mean (SEM)) 24 (2) Omega cm(2) in controls to 10 (1) Omega cm(2). Mannitol flux increased from 113 (24) nmol h(-1) cm(-2) in controls to 242 (29) nmol h(-1) cm(-2). Microdissection revealed a villus surface area reduced by 47% (6.6%). Intraepithelial lymphocytes were increased to 63 (7) per 100 enterocytes, with an increased rate of perforin-positive cytotoxic T cells. Expression of tight junctional proteins occludin, claudin-4 and claudin-5 was reduced. The epithelial apoptotic ratio was doubled in norovirus infection. Furthermore, the basal short circuit current was increased in norovirus infection and could be reduced by bumetanide and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). CONCLUSIONS: Norovirus infection leads to epithelial barrier dysfunction paralleled by a reduction of sealing tight junctional proteins and an increase in epithelial apoptosis, which may partly be mediated by increased cytotoxic intraepithelial lymphocytes. Furthermore, active anion secretion is markedly stimulated. Thus, the diarrhoea in norovirus infection is driven by both a leak flux and a secretory component.

Impairment of the intestinal barrier is evident in untreated but absent in suppressively treated HIV-infected patients.

BACKGROUND AND AIMS: Impairment of the gastrointestinal mucosal barrier contributes to progression of HIV infection. The purpose of this study was to investigate the effect of highly active antiretroviral therapy (HAART) on the HIV-induced intestinal barrier defect and to identify underlying mechanisms. METHODS: Epithelial barrier function was characterised by impedance spectroscopy and [(3)H]mannitol fluxes in duodenal biopsies from 11 untreated and 8 suppressively treated HIV-infected patients, and 9 HIV-seronegative controls. The villus/crypt ratio was determined microscopically. Epithelial apoptoses were analysed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) and caspase-3 staining. Tight junction protein expression was quantified by densitometric analysis of immunoblots. Mucosal cytokine production was determined by cytometric bead array. RESULTS: Only in untreated but not in treated HIV-infected patients, epithelial resistance was reduced (13 (1) vs 23 (2) ohm cm(2), p<0.01) and mannitol permeability was increased compared with HIV-negative controls (19 (3) vs 9 (1) nm/s, p<0.05). As structural correlates, epithelial apoptoses and expression of the pore-forming claudin-2 were increased while expression of the sealing claudin-1 was reduced in untreated compared with treated patients and HIV-negative controls. Furthermore, villous atrophy was evident and mucosal production of interleukin 2 (IL2), IL4 and tumour necrosis factor alpha (TNFalpha) was increased in untreated but not in treated HIV-infected patients. Incubation with IL2, IL4, TNFalpha and IL13 reduced the transepithelial resistance of rat jejunal mucosa. CONCLUSIONS: Suppressive HAART abrogates HIV-induced intestinal barrier defect and villous atrophy. The HIV-induced barrier defect is due to altered tight junction protein composition and elevated epithelial apoptoses. Mucosal cytokines are mediators of the HIV-induced mucosal barrier defect and villous atrophy.

TNFalpha up-regulates claudin-2 expression in epithelial HT-29/B6 cells via phosphatidylinositol-3-kinase signaling.

Our aim has been to characterize the molecular mechanisms regulating the expression of the channel-forming tight-junctional protein claudin-2 in response to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFalpha), which is elevated, for example, in active Crohn's disease. TNFalpha caused an 89% decrease of the paracellular resistance in colonic HT-29/B6 cells, whereas transcellular resistance was unaltered. The claudin-2 protein level was increased by TNFalpha without changes in subcellular tight-junctional protein localization as revealed by confocal laser scanning microscopy. Enhanced gene expression was identified as the source of this increase, since claudin-2-specific mRNA and promoter activity was elevated, whereas mRNA stability remained unaltered. Specific inhibitors and phospho-specific antibodies revealed that the increased gene expression of claudin-2 after TNFalpha treatment was mediated by the phosphatidylinositol-3-kinase pathway. Thus, the up-regulation of claudin-2 by TNFalpha is attributable to the regulation of the expression of the gene, as a result of which epithelial barrier function is disturbed, for example, during chronic intestinal inflammation.

Mechanisms of epithelial translocation of the alpha(2)-gliadin-33mer in coeliac sprue.

BACKGROUND AND AIMS: The alpha(2)-gliadin-33mer has been shown to be important in the pathogenesis of coeliac disease. We aimed to study mechanisms of its epithelial translocation and processing in respect to transcytotic and paracellular pathways. METHODS: Transepithelial passage of a fluorescence-labelled alpha(2)-gliadin-33mer was studied in Caco-2 cells by using reverse-phase high-performance liquid chromatography, mass spectrometry, confocal laser scanning microscopy (LSM) and fluorescence activated cell sorting (FACS). Endocytosis mechanisms were characterised with rab-GFP constructs transiently transfected into Caco-2 cells and in human duodenal biopsy specimens. RESULTS: The alpha(2)-gliadin-33mer dose-dependently crossed the epithelial barrier in the apical-to-basal direction. Degradation analysis revealed translocation of the 33mer polypeptide in the uncleaved as well as in the degraded form. Transcellular passage was identified by confocal LSM, inhibitor experiments and FACS. Rab5 but not rab4 or rab7 vesicles were shown to be part of the transcytotic pathway. After pre-incubation with interferon-gamma, translocation of the 33mer was increased by 40%. In mucosal biopsies of the duodenum, epithelial 33mer uptake was significantly higher in untreated coeliac disease patients than in healthy controls or coeliac disease patients on a gluten-free diet. CONCLUSION: Epithelial translocation of the alpha(2)-gliadin-33mer occurs by transcytosis after partial degradation through a rab5 endocytosis compartment and is regulated by interferon-gamma. Uptake of the 33mer is higher in untreated coeliac disease than in controls and coeliac disease patients on a gluten-free diet.

Tricellulin is regulated via interleukin-13-receptor α2, affects macromolecule uptake, and is decreased in ulcerative colitis.

In the two inflammatory bowel diseases, ulcerative colitis (UC) and Crohn's disease (CD), altered expression of tight junction (TJ) proteins leads to an impaired epithelial barrier including increased uptake of luminal antigens supporting the inflammation. Here, we focused on regulation of tricellulin (Tric), a protein of the tricellular TJ essential for the barrier against macromolecules, and hypothesized a role in paracellular antigen uptake. We report that Tric is downregulated in UC, but not in CD, and that its reduction increases the passage of macromolecules. Using a novel visualization method, passage sites were identified at TJ regions usually sealed by Tric. We show that interleukin-13 (IL-13), beyond its known effect on claudin-2, downregulates Tric expression. These two effects of IL-13 are regulated by different signaling pathways: The IL-13 receptor α1 upregulates claudin-2, whereas IL-13 receptor α2 downregulates Tric. We suggest to target the α2 receptor in future developments of therapeutical IL-13-based biologicals.

Campylobacter jejuni impairs sodium transport and epithelial barrier function via cytokine release in human colon.

This corrects the article DOI: 10.1038/mi.2017.66.

Campylobacter jejuni impairs sodium transport and epithelial barrier function via cytokine release in human colon.

Campylobacter jejuni is the most prevalent cause of foodborne bacterial enteritis worldwide. Patients present with diarrhea and immune responses lead to complications like arthritis and irritable bowel syndrome. Although studies exist in animal and cell models, we aimed at a functional and structural characterization of intestinal dysfunction and the involved regulatory mechanisms in human colon. First, in patients' colonic biopsies, sodium malabsorption was identified as an important diarrheal mechanism resulting from hampered epithelial ion transport via impaired epithelial sodium channel (ENaC) ß- and γ-subunit. In addition, barrier dysfunction from disrupted epithelial tight junction proteins (claudin-1, -3, -4, -5, and -8), epithelial apoptosis, and appearance of lesions was detected, which cause leak-flux diarrhea and can perpetuate immune responses. Importantly, these effects in human biopsies either represent direct action of Campylobacter jejuni (ENaC impairment) or are caused by proinflammatory signaling (barrier dysfunction). This was revealed by regulator analysis from RNA-sequencing (cytometric bead array-checked) and confirmed in cell models, which identified interferon-γ, TNFα, IL-13, and IL-1ß. Finally, bioinformatics' predictions yielded additional information on protective influences like vitamin D, which was confirmed in cell models. Thus, these are candidates for intervention strategies against C. jejuni infection and post-infectious sequelae, which result from the permissive barrier defect along the leaky gut.

Complex phenotype of mice lacking occludin, a component of tight junction strands.

Occludin is an integral membrane protein with four transmembrane domains that is exclusively localized at tight junction (TJ) strands. Here, we describe the generation and analysis of mice carrying a null mutation in the occludin gene. Occludin -/- mice were born with no gross phenotype in the expected Mendelian ratios, but they showed significant postnatal growth retardation. Occludin -/- males produced no litters with wild-type females, whereas occludin -/- females produced litters normally when mated with wild-type males but did not suckle them. In occludin -/- mice, TJs themselves did not appear to be affected morphologically, and the barrier function of intestinal epithelium was normal as far as examined electrophysiologically. However, histological abnormalities were found in several tissues, i.e., chronic inflammation and hyperplasia of the gastric epithelium, calcification in the brain, testicular atrophy, loss of cytoplasmic granules in striated duct cells of the salivary gland, and thinning of the compact bone. These phenotypes suggested that the functions of TJs as well as occludin are more complex than previously supposed.

Claudin-2-mediated cation and water transport share a common pore.

AIM: Claudin-2 is a tight junction protein typically located in 'leaky' epithelia exhibiting large paracellular permeabilities like small intestine and proximal kidney tubule. Former studies revealed that claudin-2 forms paracellular channels for small cations like sodium and potassium and also paracellular channels for water. This study analyses whether the diffusive transport of sodium and water occurs through a common pore of the claudin-2 channel. METHODS: Wild-type claudin-2 and different claudin-2 mutants were expressed in MDCK I kidney tubule cells using an inducible system. Ion and water permeability and the effect of blocking reagents on both were investigated on different clones of the mutants. RESULTS: Neutralization of a negatively charged cation interaction site in the pore with the mutation, D65N, decreased both sodium permeability and water permeability. Claudin-2 mutants (I66C and S68C) with substitution of the pore-lining amino acids with cysteine were used to test the effect of steric blocking of the claudin-2 pore by thiol-reactive reagents. Addition of thiol-reactive reagents to these mutants simultaneously decreased conductance and water permeability. Remarkably, all experimental perturbations caused parallel changes in ion conductance and water permeability, disproving different or independent passage pathways. CONCLUSION: Our results indicate that claudin-2-mediated cation and water transport are frictionally coupled and share a common pore. This pore is lined and determined in permeability by amino acid residues of the first extracellular loop of claudin-2.

Architectural and functional alterations of the small intestinal mucosa in classical Whipple's disease.

Classical Whipple's disease (CWD) affects the gastrointestinal tract and rather elicits regulatory than inflammatory immune reactions. Mechanisms of malabsorption, diarrhea, and systemic immune activation are unknown. We here analyzed mucosal architecture, barrier function, and immune activation as potential diarrheal trigger in specimens from 52 CWD patients. Our data demonstrate villus atrophy and crypt hyperplasia associated with epithelial apoptosis and reduced alkaline phosphatase expression in the duodenum of CWD patients. Electrophysiological and flux experiments revealed increased duodenal permeability to small solutes and macromolecules. Duodenal architecture and permeability ameliorated upon antibiotic treatment. Structural correlates for these alterations were concordant changes of membranous claudin-1, claudin-2, claudin-3, and tricellulin expression. Tumor necrosis factor-α and interleukin-13 were identified as probable mediators of epithelial apoptosis, and altered tight junction expression. Increased serum markers of microbial translocation and their decline following treatment corroborated the biological significance of the mucosal barrier defect. Hence, mucosal immune responses in CWD elicit barrier dysfunction. Diarrhea is caused by loss of absorptive capacity and leak flux of ions and water. Downregulation of tricellulin causes increased permeability to macromolecules and subsequent microbial translocation contributes to systemic inflammation. Thus, therapeutic strategies to reconstitute the mucosal barrier and control inflammation could assist symptomatic control of CWD.

Epithelial transport and barrier function in occludin-deficient mice.

BACKGROUND AND AIMS: This study aimed at functional characterization of the tight junction protein occludin using the occludin-deficient mouse model. METHODS: Epithelial transport and barrier functions were characterized in Ussing chambers. Impedance analysis revealed the ionic permeability of the epithelium (Re, epithelial resistance). Conductance scanning differentiated transcellular (Gc) and tight junctional conductance (Gtj). The pH-stat technique quantified gastric acid secretion. RESULTS: In occludin+/+ mice, Re was 23+/-5 Omega cm2 in jejunum, 66+/-5 Omega cm2 in distal colon and 33+/-6 Omega cm2 in gastric corpus and was not altered in heterozygotic occludin+/- or homozygotic occludin-/- mice. Additionally, [3H]mannitol fluxes were unaltered. In the control colon, Gc and Gtj were 7.6+/-1.0 and 0.3+/-0.1 mS/cm2 and not different in occludin deficiency. Epithelial resistance after mechanical perturbation or EGTA exposition (low calcium switch) was not more affected in occludin-/- mice than in control. Barrier function was measured in the urinary bladder, a tight epithelium, and in the stomach. Control Rt was 5.8+/-0.8 kOmega cm2 in urinary bladder and 33+/-6 Omega cm2 in stomach and not altered in occludin-/- mice. In gastric corpus mucosa, the glandular structure exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished in occludin-/- mice. CONCLUSION: Epithelial barrier characterization in occludin-deficiency points against an essential barrier function of occludin within the tight junction strands or to a substitutional redundancy of single tight junction molecules like occludin. A dramatic change in gastric morphology and secretory function indicates that occludin is involved in gastric epithelial differentiation.

pH dependence of protamine action on apical membrane permeability in Necturus gallbladder epithelium.

Protamine reversibly decreases cation permeability and alters the structure of Necturus gallbladder tight junctions. Conflicting results, however, have been published whether or not it also affects apical cell membrane permeability. We investigated this issue more systematically by measuring voltage (psi mc) and fractional resistance (fRa) of the apical membrane at varying concentrations of protamine, K+, and H+ in the bathing solution. At pH 7.6 and [K+] 2.5 mM, (Poler, M.S. and Reuss, L. (1987) Am. J. Physiol. 253, C662) 6 microM protamine caused psi mc to depolarize from -58 to -51 mV and fRa to decrease from 0.74 to 0.67. If we increased pH to 8.1 these effects were even more pronounced. At [K+] 2.5 mM, but not 4.5 mM, psi mc transiently hyperpolarized for about 5 min after adding protamine. Most importantly, if [K+] was 4.5 mM and pH was adjusted to 7.1 (Bentzel et al. (1987) J. Membr. Biol. 95, 9) no significant changes of psi mc and fRa occurred. In any case, at a supramaximal concentration of 200 microM, protamine did not further increase the paracellular response but produced decreasing psi mc and fRa. We conclude that 6 microM protamine decreases K+ conductance of the apical membrane, if it is already tuned high by high pH. At low control K+ conductance as observed at lower pH, protamine action is restricted to the paracellular pathway. Thus, conflicting results were due to different experimental conditions. At a solution pH of 7.1, 6 microM protamine fulfills criteria of a selective tool for reversibly altering structure and function of the tight junction in Necturus gallbladder.

Claudin-2 as a mediator of leaky gut barrier during intestinal inflammation.

The epithelial tight junction determines the paracellular water and ion movement in the intestine and also prevents uptake of larger molecules, including antigens, in an uncontrolled manner. Claudin-2, one of the 27 mammalian claudins regulating that barrier function, forms a paracellular channel for small cations and water. It is typically expressed in leaky epithelia like proximal nephron and small intestine and provides a major pathway for the paracellular transport of sodium, potassium, and fluid. In intestinal inflammation (Crohn's disease, ulcerative colitis), immune-mediated diseases (celiac disease), and infections (HIV enteropathy), claudin-2 is upregulated in small and large intestine and contributes to diarrhea via a leak flux mechanism. In parallel to that upregulation, other epithelial and tight junctional features are altered and the luminal uptake of antigenic macromolecules is enhanced, for which claudin-2 may be partially responsible through induction of tight junction strand discontinuities.

Duodenal biopsies of HIV-infected patients with diarrhoea exhibit epithelial barrier defects but no active secretion.

OBJECTIVES: To characterize diarrhoeal mechanisms in HIV-infected patients, epithelial transport and barrier function of the duodenal mucosa was investigated in vitro. PATIENTS: Twenty-one HIV-seropositive patients (13 asymptomatic and eight with diarrhoea) and 12 controls from an urban referral-based tertiary care centre in Berlin who underwent duodenoscopy. METHODS: A new miniaturized Ussing chamber allowed measurements on duodenal forceps biopsies. Epithelial barrier function was characterized by alternating current impedance analysis, which allows differentiation of epithelial and subepithelial resistance and by 3H-lactulose and 3H-mannitol flux measurements. Na+-glucose cotransport was quantified as phlorizin-sensitive short circuit current (Isc) and active ion secretion by baseline and bumetanide-sensitive Isc. RESULTS: Duodenal biopsies from asymptomatic HIV-infected patients were no different from controls, whereas biopsies from HIV-infected patients with diarrhoea showed a decrease in epithelial resistance from 21.2+/-1.9 to 12.9+/-1.3 omega cm2 (P<0.01). Concomitantly, mucosal-to-serosal lactulose flux increased from 0.29+/-0.02 to 0.40+/-0.03 micromol (hcm2) (P<0.01). Phlorizin-sensitive Isc indicating Na+-glucose cotransport, as well as baseline and bumetanide-sensitive Isc indicating active electrogenic chloride secretion were not different between the three groups. CONCLUSIONS: A miniaturized Ussing device was developed for electrophysiological investigations of duodenal forceps biopsies, which allowed characterization of active ion transport mechanisms and epithelial barrier function. Duodenum of HIV-infected patients with diarrhoea showed no evidence for active ion secretion or Na+-glucose malabsorption, but showed an impaired epithelial barrier function, which could contribute to diarrhoea by a leak flux mechanism.

Mucosal abnormalities in microsporidiosis.

OBJECTIVE: To determine the prevalence of microsporidiosis in HIV-infected patients with and without diarrhoea and to characterize alterations in mucosal architecture and brush border enzyme activities in patients with microsporidiosis. PATIENTS: A total of 259 HIV-infected patients undergoing oesophago-gastroduodenoscopy because of diarrhoea (n = 123) or other symptoms (n = 136) were studied. METHODS: Patients were evaluated for the presence of microsporidia by electron microscopy of duodenal biopsies. Brush border enzyme activities were measured by histochemistry and mucosal architecture was determined by three-dimensional morphometry in biopsies from patients with microsporidiosis and compared with biopsies from a subgroup of HIV-infected patients with or without other enteropathogens. RESULTS: Enterocytozoon bieneusi was detected in 17 patients and Encephalitozoon intestinalis was detected in two patients. Microsporidiosis was significantly more frequent in patients with chronic diarrhoea (19.1%; P < 0.0001) or in patients with acute diarrhoea (7.2%; P = 0.04) than in patients without diarrhoea (1.5%). Microsporidiosis was associated with lactase deficiency (P = 0.03) and a reduced activity of alkaline phosphatase (P = 0.028) and alpha-glucosidase (P = 0.025) at the basal part of the villus compared with brush border enzymes in patients without enteropathogens. Patients with microsporidia had reduced villus height (P = 0.043) and a villus surface reduced by 40% (P = 0.004) compared with patients with enteropathogens other than microsporidia. CONCLUSIONS: Our study confirms the association between microsporidia and diarrhoea. The pathophysiologic mechanism by which microsporidia cause diarrhoea appears in part to be malabsorption, caused by a reduction of absorptive mucosal surface and impairment of enterocyte function.