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.

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.

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.

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.

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.

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.

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.

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.

TcpC protein from E. coli Nissle improves epithelial barrier function involving PKCζ and ERK1/2 signaling in HT-29/B6 cells.

The probiotic Escherichia coli Nissle 1917 (EcN) is widely used to maintain remission in ulcerative colitis. This is thought to be mediated by various immunomodulatory and barrier-stabilizing effects in the intestine. In this study, the mechanisms of barrier modulation by EcN were studied in the human epithelial HT-29/B6 cell culture model.EcN supernatant increased transepithelial resistance (TER) and reduced permeability to mannitol because of sealing of the paracellular passage pathway as revealed by two-path impedance spectroscopy. This increase in TER was attributed to the TcpC protein of EcN. TcpC induced protein kinase C-ζ (PKCζ) and extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation, which in turn resulted in upregulation of the barrier-forming tight junction protein claudin-14. By specific silencing of protein expression by small interfering RNA (siRNA), the sealing function of claudin-14 was confirmed. In conclusion, the TcpC protein of EcN affects innate immunity by improving intestinal barrier function through upregulation of claudin-14 via PKCζ and ERK1/2 signaling.

Increased intestinal permeability in malnourished patients with liver cirrhosis.

BACKGROUND/OBJECTIVE: Malnutrition is a prominent feature in liver cirrhosis, with deleterious impact on clinical outcome. The objective of this study is to investigate whether malnutrition is associated with increased gastrointestinal permeability in liver cirrhosis reflected by altered urinary excretion of non-metabolizable sugar probes. SUBJECTS/METHODS: Patients with advanced liver cirrhosis (Child Pugh Score B or C) were recruited. Nutritional status was determined according to the Subjective Global Assessment. Intestinal permeability was assessed by measuring the urinary excretion of orally administered, non-metabolized sugar probe molecules. The lactulose/mannitol ratio served as marker for intestinal permeability and reflects non-carrier-mediated transcellular and paracellular transport of the small intestine during the first 5 h. Sucrose recovery in urine within the first 5 h reflects gastroduodenal permeability; sucralose recovery in urine 5-26 h after consumption reflects colonic permeability. RESULTS: Sixty-four patients (56.7±10.8 years; 33% female) were included in the study. Twenty-one patients were considered well nourished according to the Subjective Global Assessment, 23 moderately nourished and 20 patients severely malnourished; 74% had alcoholic liver disease and 67% had cirrhosis stage Child C. Gastroduodenal and colonic permeability was significantly increased in patients with liver cirrhosis compared with 63 healthy controls (0.23±0.22 and 1.37±1.42% vs 0.14±0.10 and 0.41±0.72% in controls), but not different between well and malnourished subjects. Small intestinal permeability (lactulose/mannitol ratio) was increased in all patients (0.069±0.055%) and further increased in malnourished patients (0.048±0.031% vs 0.084±0.061%, P=0.004) due to decreased mannitol recovery only. CONCLUSIONS: Gastric, small intestinal and even colonic permeability was altogether increased in liver cirrhosis, and malnutrition was associated with further increased small intestinal permeability indicative of villous atrophy.

Mesenteric fat - control site for bacterial translocation in colitis?

In Crohn's disease bacteria could be detected in the adjacent mesenteric fat characterized by hypertrophy of unknown function. This study aimed to define effector responses of this compartment induced by bacterial translocation during intestinal inflammation. Dextran sulfate sodium-induced colitis served as a model of intestinal inflammation. Translocation of peptides and bacteria into mesenteric fat was evaluated. Innate functions of mesenteric fat and epithelium were characterized at whole tissue, cellular, and effector molecule levels. Orally applied peptides translocated in healthy wild-type (WT) mice. Bacterial translocation was not detected in healthy and acute but increased in chronic colitis. Mesenteric fat from colitic mice released elevated levels of cytokines and was infiltrated by immune cells. In MyD88(-/-) mice bacterial translocation occurred in health and increased in colitis. The exaggerated cytokine production in mesenteric fat accompanying colonic inflammation in WT mice was less distinct in MyD88(-/-) mice. In vitro studies revealed that fat not only increases cytokine production following contact with bacterial products, but also that preadipocytes are potent phagocytes. Colonic inflammation is accompanied by massive cytokine production and immune cell infiltration in adjacent adipose tissue. These effects can be considered as protective mechanisms of the mesenteric fat in the defense of bacterial translocation.

CCR7 deficiency causes diarrhea associated with altered ion transport in colonocytes in the absence of overt colitis.

The chemokine receptor CCR7 is a central regulator in the maintenance of cellular homeostasis of mucosal tissues. CCR7⁻/⁻ mice develop autoimmune gastritis and exocrinopathy accompanied by the formation of mucosal tertiary lymphoid follicles. Here we found that CCR7-deficient mice frequently suffered from chronic diarrhea linked with increased gastrointestinal motility and the development of severe anorectal prolapse. Enhanced formation of intestinal lymphoid follicles was associated with an elevated proportion of activated colonic T cells and increased production of the cytokine interleukin (IL)-1ß. To uncover the pathomechanisms of diarrhea in CCR7⁻/⁻ mice, colonic epithelial barrier and ion channel activities were analyzed in Ussing chambers. Although overt acute colitis was absent, CCR7 deficiency resulted in reduced electrogenic sodium absorption and colonic chloride secretion. As it is known that IL-1ß regulates epithelial sodium channel (ENaC) activity, these data imply a causal link between CCR7 expression, IL-1ß level, and Na⁺ malabsorption owing to altered ENaC expression and diarrhea.

Cost-effectiveness of a 3-month intervention with oral nutritional supplements in disease-related malnutrition: a randomised controlled pilot study.

BACKGROUND/OBJECTIVES: Nutritional intervention with oral nutritional supplements (ONS) has been shown to increase quality of life in malnourished patients. We investigated whether post-hospital supplementation with ONS is cost-effective according to international benchmarks in malnourished patients. SUBJECTS/METHODS: In total, 114 malnourished patients (50.6±16.1 years, 57 female) with benign gastrointestinal disease were included and randomised to receive either ONS for 3 months and dietary counselling at discharge (intervention, n=60) or only dietary counselling at discharge (control group, n=54). Nutritional status was assessed with Subjective Global Assessment. Intervention patients documented daily intake of ONS; quality of life was assessed with Short-Form (SF)-36 Health Survey and SF-36 values were transformed into health-status utilities. Quality-adjusted life years (QALYs) were calculated by adopting the area under the curve method. We used two different pricing scenarios for ONS (minimum price: [euro]2.30 and maximum: [euro]2.93/tetrapack). The incremental cost-effectiveness ratio (ICER) of supplementation with ONS was calculated for both price scenarios. All analyses were corrected for age and gender. RESULTS: Intervention patients consumed 2.4±0.8 ONS per day. Intervention and control patients did not differ in their health status utilities at baseline (0.594±0.017 vs 0.619±0.018), but after 3 months, the health status utilities were significantly higher in intervention patients than in control patients (0.731±0.015 vs 0.671±0.016, P=0.028). Intervention was associated with significantly higher costs (ICER: [euro]9497 and [euro]12,099/additional QALY, respectively) but deemed cost-effective according to international thresholds (< [euro]50,000/QALY). CONCLUSIONS: A 3-month intervention with ONS increases quality of life in malnourished patients. This treatment appears to be cost-effective according to international benchmarks.

Prostaglandin I(2) sensory input into the enteric nervous system during distension-induced colonic chloride secretion in rat colon.

AIM: Intestinal pressure differences or experimental distension induce ion secretion via the enteric nervous system, the sensorial origin of which is only poorly understood. This study aimed to investigate sensorial inputs and the role of afferent and interneurones in mechanically activated submucosal secretory reflex circuits. METHODS: Distension-induced rheogenic chloride secretion was measured as increase in short-circuit current 10 min after distension (DeltaI(SC)(10); distension parameters +/- 100 microL, 2 Hz, 20 s) in partially stripped rat distal colon in the Ussing-chamber in vitro. PGE(2) and PGI(2) were measured by radioimmunoassay. RESULTS: DeltaI(SC)(10) was 2.0 +/- 0.2 micromol h(-1) cm(-2) and could be attenuated by lobeline, mecamylamine and dimethylphenylpiperazine, indicating an influence of nicotinergic interneurones. Additionally, a contribution of afferent neurones was indicated from the short-term potentiation of DeltaI(SC)(10) by capsaicin (1 microm). As evidence for its initial event, indomethacin (1 microm) inhibited distension-induced secretion and the release of PGI(2) was directly detected after distension. Furthermore, serotoninergic mediation was confirmed by granisetron (100 microm) which was functionally localized distally to PGI(2) in this reflex circuit, as granisetron inhibited an iloprost-induced I(SC), while indomethacin did not affect serotonin-activated ion secretion. CONCLUSIONS: Distension-induced active electrogenic chloride secretion in rat colon is mediated by a neuronal reflex circuit which includes afferent neurones and nicotinergic interneurones. It is initiated by distension-induced PGI(2) release from subepithelial cells triggering this reflex via serotoninergic 5-HT(3) receptor transmission. Functionally, this mechanism may help to protect against intestinal stasis but could also contribute to luminal fluid loss, e.g. during intestinal obstruction.

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.

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.