Hanno Troeger

Epithelial Tight Junctions in Intestinal Inflammation

The epithelium in inflamed intestinal segments of patients with Crohns disease is characterized by a reduction of tight junction strands, strand breaks, and alterations of tight junction protein content and composition. In ulcerative colitis, epithelial leaks appear early due to micro‐erosions resulting from upregulated epithelial apoptosis and in addition to a prominent increase of claudin‐2. Th1‐cytokine effects by interferon‐γ in combination with TNFα are important for epithelial damage in Crohns disease, while interleukin‐13 (IL‐13) is the key effector cytokine in ulcerative colitis stimulating apoptosis and upregulation of claudin‐2 expression. Focal lesions caused by apoptotic epithelial cells contribute to barrier disturbance in IBD by their own conductivity and by confluence toward apoptotic foci or erosions. Another type of intestinal barrier defect can arise from α‐hemolysin harboring E. coli strains among the physiological flora, which can gain pathologic relevance in combination with proinflammatory cytokines under inflammatory conditions. On the other hand, intestinal barrier impairment can also result from transcellular antigen translocation via an initial endocytotic uptake into early endosomes, and this is intensified by proinflammatory cytokines as interferon‐γ and may thus play a relevant role in the onset of IBD. Taken together, barrier defects contribute to diarrhea by a leak flux mechanism (e.g., in IBD) and can cause mucosal inflammation by luminal antigen uptake. Immune regulation of epithelial functions by cytokines may cause barrier dysfunction not only by tight junction impairments but also by apoptotic leaks, transcytotic mechanisms, and mucosal gross lesions.

Effect of chronic Giardia lamblia infection on epithelial transport and barrier function in human duodenum

Background:Giardia lamblia causes infection of the small intestine, which leads to malabsorption and chronic diarrhoea. Aim: To characterise the inherent pathomechanisms of G lamblia infection. Methods: Duodenal biopsy specimens from 13 patients with chronic giardiasis and from controls were obtained endoscopically. Short-circuit current (ISC) and mannitol fluxes were measured in miniaturised Ussing chambers. Epithelial and subepithelial resistances were determined by impedance spectroscopy. Mucosal morphometry was performed and tight junction proteins were characterised by immunoblotting. Apoptotic ratio was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling staining. Results: In giardiasis, mucosal surface area per unit serosa area was decreased to 75% (3%) of control, as a result of which epithelial resistance should increase. Instead, epithelial resistance of giardiasis biopsy specimens was decreased (19 (2) vs 25 (2) Ω cm2; p<0.05) whereas mannitol flux was not significantly altered (140 (27) vs 105 (16) nmol/h/cm2). As structural correlate, reduced claudin 1 expression and increased epithelial apoptosis were detected. Furthermore, basal ISC increased from 191 (20) in control to 261 (12) µA/h/cm2 in giardiasis. The bumetanide-sensitive portion of ISC in giardiasis was also increased (51 (5) vs 20 (9) µA/h/cm2 in control; p<0.05). Finally, phlorizin-sensitive Na+–glucose symport was reduced in patients with giardiasis (121 (9) vs 83 (14) µA/h/cm2). Conclusions:G lamblia infection causes epithelial barrier dysfunction owing to down regulation of the tight junction protein claudin 1 and increased epithelial apoptoses. Na+-dependent d-glucose absorption is impaired and active electrogenic anion secretion is activated. Thus, the mechanisms of diarrhoea in human chronic giardiasis comprise leak flux, malabsorptive and secretory components.

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 [3H]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) Ω cm2, 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 α (TNFα) was increased in untreated but not in treated HIV-infected patients. Incubation with IL2, IL4, TNFα 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.

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) Ω cm2 in controls to 10 (1) Ω cm2. 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.

Cell polarity-determining proteins Par-3 and PP-1 are involved in epithelial tight junction defects in coeliac disease

Background Epithelial barrier defects are well known in coeliac disease, but the mechanisms are only poorly defined. It is unclear, whether barrier disturbance reflects upregulated epithelial transcytosis or paracellular leakage. Objective To characterise the molecular structure and function of the epithelial tight junction (TJ) and mechanisms of its dysregulation. Methods Molecular analysis of proteins involved in TJ assembly and their regulation was performed by western blotting and confocal microscopy correlated to electrophysiology. Results A complex alteration of the composition of epithelial TJ proteins (with more pore-forming claudins like claudin-2 and a reduction in tightening claudins like claudin-3, -5 and -7) was found for protein expression and subcellular localisation, responsible for an increase in paracellular biotin-NHS uptake. In contrast, epithelial apoptosis was only moderately elevated (accounting for a minor portion of barrier defects) and epithelial gross lesions—for example, at cell extrusion zones, were absent. This TJ alteration was linked to an altered localisation/expression of proteins regulating TJ assembly, the polarity complex protein Par-3 and the serine-/threonine phosphatase PP-1. Conclusions Changes in cell polarity proteins Par-3 and PP-1 are associated with altered expression and assembly of TJ proteins claudin-2, -3, -5 and -7 and ZO-1, causing paracellular leakage in active coeliac disease.

Arcobacter butzleri Induces Barrier Dysfunction in Intestinal HT-29/B6 Cells

BACKGROUND Arcobacter butzleri causes watery diarrhea and bacteremia. Although, recently, more cases of diarrhea have been caused by Arcobacter species, very little is known about its pathogenesis, the identification of which is the aim of this study. METHODS Human HT-29/B6 colonic epithelial monolayers were apically inoculated with A. butzleri. Transepithelial resistance and macromolecule fluxes were measured in Ussing chambers. Tight junction protein expression was analyzed by Western blotting, and subcellular distribution was analyzed by confocal laser-scanning microscopy. RESULTS Infection of HT-29/B6 caused a decrease in transepithelial resistance to 30% and an increase in paracellular permeability to fluorescein (10.8+/-3.5 10(-6) cm/s vs. 1.8+/-0.6 10(-6) cm/s in control; P<.05) and dextran-4 kDa (0.036+/-0.005 10(-6) cm/s vs. 0.015+/-0.002 10(-6) cm/s in control; P<.01). This effect was time and dose dependent and was also caused by bacterial lysates showing heat and proteinase-K sensitivity. As structural correlate, expression of the tight junctional proteins claudin-1, -5, and -8 was reduced, and claudin-1 and -8 were redistributed off the tight junctional strands forming intracellular aggregates. Furthermore, A. butzleri induced epithelial apoptosis (3-fold). CONCLUSIONS A. butzleri induces epithelial barrier dysfunction by changes in tight junction proteins and induction of epithelial apoptosis, which are mechanisms that are consistent with a leak flux type of diarrhea in A. butzleri infection.

Escherichia coli alpha-haemolysin induces focal leaks in colonic epithelium: a novel mechanism of bacterial translocation

Extraintestinal pathogenic Escherichia coli (ExPEC) are usually harmless colonizer of the intestinal microflora. However, they are capable to translocate and cause life‐threatening disease. Translocation of ExPEC isolates was quantified in colonic monolayers. Transepithelial resistance (Rt) was monitored and local changes in conductivity analysed with conductance scanning. Confocal microscopy visualized the translocation route. Corroboratory experiments were performed on native rat colon. One translocating strain E. coli O4 was identified. This translocation process was associated with an Rt decrease (36 ± 1% of initial resistance) beginning only 2 h after inoculation. The sites of translocation were small defects in epithelial integrity (focal leaks) exhibiting highly increased local ion permeability. Translocation was enhanced by preincubation of monolayers with tumour necrosis factor‐α or interleukin‐13. Mutant strains lacking alpha‐haemolysin lost the ability to induce focal leaks, while this effect could be restored by re‐introducing the haemolysin determinant. Filtrate of a laboratory strain carrying the alpha‐haemolysin operon was sufficient for focal leak induction. In native rat colon, E. coli O4 decreased Rt and immunohistology demonstrated focal leaks resembling those in cell monolayers. E. coliα‐haemolysin is able to induce focal leaks in colonic cell cultures as well as in native colon. This process represents a novel route of bacterial translocation facilitated by pro‐inflammatory cytokines.

Enterotoxicity of a nonribosomal peptide causes antibiotic-associated colitis

Significance The human gut microbiota is a complex community of microbes with enormous metabolic potential. Recognition of the significance of bacterial metabolites in mediating host interactions and the impact of perturbations of this ecosystem on human health has increased dramatically. Antibiotic therapy eliminates not only pathogens but also some of the commensal enteric microbiota, sometimes leading to inflammation and diarrhea. Understanding how microbial imbalance actually causes disease is challenging. This study reveals how a gut resident is able to cause colitis during penicillin therapy. We show that a pyrrolobenzodiazepine metabolite produced by Klebsiella oxytoca directly damages the intestinal epithelium and disrupts its protective barrier function. The enterotoxicity of tilivalline provides a mechanism for antibiotic-induced colitis. Antibiotic therapy disrupts the human intestinal microbiota. In some patients rapid overgrowth of the enteric bacterium Klebsiella oxytoca results in antibiotic-associated hemorrhagic colitis (AAHC). We isolated and identified a toxin produced by K. oxytoca as the pyrrolobenzodiazepine tilivalline and demonstrated its causative action in the pathogenesis of colitis in an animal model. Tilivalline induced apoptosis in cultured human cells in vitro and disrupted epithelial barrier function, consistent with the mucosal damage associated with colitis observed in human AAHC and the corresponding animal model. Our findings reveal the presence of pyrrolobenzodiazepines in the intestinal microbiota and provide a mechanism for colitis caused by a resident pathobiont. The data link pyrrolobenzodiazepines to human disease and identify tilivalline as a target for diagnosis and neutralizing strategies in prevention and treatment of colitis.

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.

α-Haemolysin of Escherichia coli in IBD: a potentiator of inflammatory activity in the colon

Objective α-Haemolysin (HlyA) influences host cell ionic homeostasis and causes concentration-dependent cell lysis. As a consequence, HlyA-producing Escherichia coli is capable of inducing ‘focal leaks’ in colon epithelia, through which bacteria and antigens translocate. This study addressed the role of HlyA as a virulence factor in the pathogenesis of colitis according to the ‘leaky gut’ concept. Design To study the action of HlyA in the colon, we performed oral administration of HlyA-expressing E coli-536 and its isogenic α-haemolysin-deficient mutant (HDM) in three mouse models: wild type, interleukin-10 knockout mice (IL-10−/−) and monoassociated mice. Electrophysiological properties of the colonised colon were characterised in Ussing experiments. Inflammation scores were evaluated and focal leaks in the colon were assessed by confocal laser-scanning microscopy. HlyA quantity in human colon biopsies was measured by quantitative PCR. Results All three experimental mouse models infected with HlyA-producing E coli-536 showed an increase in focal leak area compared with HDM. This was associated with a decrease in transepithelial electrical resistance and an increase in macromolecule uptake. As a consequence, inflammatory activity index was increased to a higher degree in inflammation-prone mice. Mucosal samples from human colon were E coli HlyA-positive in 19 of 22 patients with ulcerative colitis, 9 of 9 patients with Crohns disease and 9 of 12 healthy controls. Moreover, focal leaks were found together with 10-fold increased levels of HlyA in active ulcerative colitis. Conclusions E coli HlyA impairs intestinal barrier function via focal leak induction in the epithelium, thereby intensifying antigen uptake and triggering intestinal inflammation in vulnerable mouse models. Therefore, HlyA-expressing E coli strains should be considered as potential cofactors in the pathogenesis of intestinal inflammation.