Diarrheal Mechanisms and the Role of Intestinal Barrier Dysfunction in Campylobacter Infections.

Campylobacter enteritis is the most common cause of foodborne bacterial diarrhea in humans. Although various studies have been performed to clarify the pathomechanism in Campylobacter infection, the mechanism itself and bacterial virulence factors are yet not completely understood. The purpose of this chapter is to (i) give an overview on Campylobacter-induced diarrheal mechanisms, (ii) illustrate underlying barrier defects, (iii) explain the role of the mucosal immune response and (iv) weigh preventive and therapeutic approaches. Our present knowledge of pathogenetic and diarrheal mechanisms of Campylobacter jejuni is explained in the first part of this chapter. In the second part, the molecular basis for the Campylobacter-induced barrier dysfunction is compared with that of other species in the Campylobacter genus. The bacteria are capable of overcoming the intestinal epithelial barrier. The invasion into the intestinal mucosa is the initial step of the infection, followed by a second step, the epithelial barrier impairment. The extent of the impairment depends on various factors, including tight junction dysregulation and epithelial apoptosis. The disturbed intestinal epithelium leads to a loss of water and solutes, the leak flux type of diarrhea, and facilitates the uptake of harmful antigens, the leaky gut phenomenon. The barrier dysfunction is accompanied by increased pro-inflammatory cytokine secretion, which is partially responsible for the dysfunction. Moreover, cytokines also mediate ion channel dysregulation (e.g., epithelial sodium channel, ENaC), leading to another diarrheal mechanism, which is sodium malabsorption. Future perspectives of Campylobacter research are the clarification of molecular pathomechanisms and the characterization of therapeutic and preventive compounds to combat and prevent Campylobacter infections.

Immune-Mediated Aggravation of the Campylobacter concisus-Induced Epithelial Barrier Dysfunction.

Campylobacter concisus is a human-pathogenic bacterium of the gastrointestinal tract. This study aimed at the contribution of the mucosal immune system in the context of intestinal epithelial barrier dysfunction induced by C. concisus. As an experimental leaky gut model, we used in vitro co-cultures of colonic epithelial cell monolayers (HT-29/B6-GR/MR) with M1-macrophage-like THP-1 cells on the basal side. Forty-eight hours after C. concisus infection, the decrease in the transepithelial electrical resistance in cell monolayers was more pronounced in co-culture condition and 22 ± 2% (p < 0.001) higher than the monoculture condition without THP-1 cells. Concomitantly, we observed a reduction in the expression of the tight junction proteins occludin and tricellulin. We also detected a profound increase in 4 kDa FITC-dextran permeability in C. concisus-infected cell monolayers only in co-culture conditions. This is explained by loss of tricellulin from tricellular tight junctions (tTJs) after C. concisus infection. As an underlying mechanism, we observed an inflammatory response after C. concisus infection through pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) released from THP-1 cells in the co-culture condition. In conclusion, the activation of subepithelial immune cells exacerbates colonic epithelial barrier dysfunction by C. concisus through tricellulin disruption in tTJs, leading to increased antigen permeability (leaky gut concept).

Vitamin D Reverses Disruption of Gut Epithelial Barrier Function Caused by Campylobacter jejuni.

Infections by the zoonotic foodborne bacterium Campylobacter jejuni (C. jejuni) are among the most frequent causes of bacterial gastroenteritis worldwide. The aim was to evaluate the relationship between epithelial barrier disruption, mucosal immune activation, and vitamin D (VD) treatment during C. jejuni infection, using intestinal epithelial cells and mouse models focused on the interaction of C. jejuni with the VD signaling pathway and VD treatment to improve C. jejuni-induced barrier dysfunction. Our RNA-Seq data from campylobacteriosis patients demonstrate inhibition of VD receptor (VDR) downstream targets, consistent with suppression of immune function. Barrier-preserving effects of VD addition were identified in C. jejuni-infected epithelial cells and IL-10-/- mice. Furthermore, interference of C. jejuni with the VDR pathway was shown via VDR/retinoid X receptor (RXR) interaction. Paracellular leakiness of infected epithelia correlated with tight junction (TJ) protein redistribution off the TJ domain and apoptosis induction. Supplementation with VD reversed barrier impairment and prevented inhibition of the VDR pathway, as shown by restoration of transepithelial electrical resistance and fluorescein (332 Da) permeability. We conclude that VD treatment restores gut epithelial barrier functionality and decreases bacterial transmigration and might, therefore, be a promising compound for C. jejuni treatment in humans and animals.

Campylobacter concisus Impairs Sodium Absorption in Colonic Epithelium via ENaC Dysfunction and Claudin-8 Disruption.

The epithelial sodium channel (ENaC) can increase the colonic absorptive capacity for salt and water. Campylobacter concisus is a common pathogenic epsilonproteobacterium, causing enteritis and diarrhea. It can induce barrier dysfunction in the intestine, but its influence on intestinal transport function is still unknown. Therefore, our study aimed to characterize C. concisus effects on ENaC using the HT-29/B6-GR/MR (epithelial cell line HT-29/B6 transfected with glucocorticoid and mineralocorticoid receptors) cell model and mouse colon. In Ussing chambers, C. concisus infection inhibited ENaC-dependent Na+ transport as indicated by a reduction in amiloride-sensitive short circuit current (-55%, n = 15, p < 0.001). This occurred via down-regulation of ß- and γ-ENaC mRNA expression and ENaC ubiquitination due to extracellular signal-regulated kinase (ERK)1/2 activation, predicted by Ingenuity Pathway Analysis (IPA). In parallel, C. concisus reduced the expression of the sealing tight junction (TJ) protein claudin-8 and induced claudin-8 redistribution off the TJ domain of the enterocytes, which facilitates the back leakage of Na+ ions into the intestinal lumen. In conclusion, C. concisus caused ENaC dysfunction via interleukin-32-regulated ERK1/2, as well as claudin-8-dependent barrier dysfunction-both of which contribute to Na+ malabsorption and diarrhea.

Tilivalline- and Tilimycin-Independent Effects of Klebsiella oxytoca on Tight Junction-Mediated Intestinal Barrier Impairment.

Klebsiella oxytoca causes antibiotic-associated hemorrhagic colitis and diarrhea. This was attributed largely to its secreted cytotoxins tilivalline and tilimycin, inductors of epithelial apoptosis. To study whether Klebsiella oxytoca exerts further barrier effects, T84 monolayers were challenged with bacterial supernatants derived from tilivalline/tilimycin-producing AHC6 or its isogeneic tilivalline/tilimycin-deficient strain Mut-89. Both preparations decreased transepithelial resistance, enhanced fluorescein and FITC-dextran-4kDa permeabilities, and reduced expression of barrier-forming tight junction proteins claudin-5 and -8. Laser scanning microscopy indicated redistribution of both claudins off the tight junction region in T84 monolayers as well as in colon crypts of mice infected with AHC6 or Mut-89, indicating that these effects are tilivalline/tilimycin-independent. Furthermore, claudin-1 was affected, but only in a tilivalline/tilimycin-dependent manner. In conclusion, Klebsiella oxytoca induced intestinal barrier impairment by two mechanisms: the tilivalline/tilimycin-dependent one, acting by increasing cellular apoptosis and a tilivalline/tilimycin-independent one, acting by weakening the paracellular pathway through the tight junction proteins claudin-5 and -8.

Curcumin Mitigates Immune-Induced Epithelial Barrier Dysfunction by Campylobacter jejuni.

Campylobacter jejuni (C. jejuni) is the most common cause of foodborne gastroenteritis worldwide. The bacteria induce diarrhea and inflammation by invading the intestinal epithelium. Curcumin is a natural polyphenol from turmeric rhizome of Curcuma longa, a medical plant, and is commonly used in curry powder. The aim of this study was the investigation of the protective effects of curcumin against immune-induced epithelial barrier dysfunction in C. jejuni infection. The indirect C. jejuni-induced barrier defects and its protection by curcumin were analyzed in co-cultures with HT-29/B6-GR/MR epithelial cells together with differentiated THP-1 immune cells. Electrophysiological measurements revealed a reduction in transepithelial electrical resistance (TER) in infected co-cultures. An increase in fluorescein (332 Da) permeability in co-cultures as well as in the germ-free IL-10-/- mouse model after C. jejuni infection was shown. Curcumin treatment attenuated the C. jejuni-induced increase in fluorescein permeability in both models. Moreover, apoptosis induction, tight junction redistribution, and an increased inflammatory response-represented by TNF-α, IL-1ß, and IL-6 secretion-was observed in co-cultures after infection and reversed by curcumin. In conclusion, curcumin protects against indirect C. jejuni-triggered immune-induced barrier defects and might be a therapeutic and protective agent in patients.

In Colon Epithelia, Clostridium perfringens Enterotoxin Causes Focal Leaks by Targeting Claudins Which are Apically Accessible Due to Tight Junction Derangement.

Clostridium perfringens enterotoxin (CPE) causes food poisoning and antibiotic-associated diarrhea. It uses some claudin tight junction proteins (eg, claudin-4) as receptors to form Ca2+-permeable pores in the membrane, damaging epithelial cells in small intestine and colon. We demonstrate that only a subpopulation of colonic enterocytes which are characterized by apical dislocation of claudins are CPE-susceptible. CPE-mediated damage was enhanced if paracellular barrier was impaired by Ca2+ depletion, proinflammatory cytokine tumor necrosis factor α, or dedifferentiation. Microscopy, Ca2+ monitoring, and electrophysiological data showed that CPE-mediated cytotoxicity and barrier disruption was limited by extent of CPE-binding. The latter was restricted by accessibility of non-junctional claudin molecules such as claudin-4 at apical membranes. Focal-leaks detected in HT-29/B6 colonic monolayers were verified for native tissue using colon biopsies. These mechanistic findings indicate how CPE-mediated effects may turn from self-limiting diarrhea into severe clinical manifestation such as colonic necrosis-if intestinal barrier dysfunction, eg, during inflammation facilitates claudin accessibility.

Enterotoxicity of a nonribosomal peptide causes antibiotic-associated 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 Affects Intestinal Barrier Function in the Colon.

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

Interleukin-13 affects the epithelial sodium channel in the intestine by coordinated modulation of STAT6 and p38 MAPK activity.

KEY POINTS: Interleukin-13 (IL-13) causes intestinal epithelial barrier dysfunction, and is implicated in the pathogenesis of Th2-driven intestinal inflammation (e.g. ulcerative colitis). However, it is unclear whether the epithelial sodium channel (ENaC) - the main limiting factor for sodium absorption in the distal colon - is also influenced by IL-13 and if so, by what mechanism(s). We demonstrate in an intestinal cell model as well as in mouse distal colon that IL-13 causes reduced ENaC activity. We show that IL-13 impairs ENaC-dependent sodium transport by activating the JAK1/2-STAT6 signalling pathway. These results improve our understanding of the mechanisms through which IL-13 functions as a key effector cytokine in ulcerative colitis, thereby contributing to the distinct pathology of this disease. ABSTRACT: Interleukin-13 (IL-13) has been strongly implicated in the pathogenesis of ulcerative colitis, possibly by disrupting epithelial integrity. In the distal colon, the epithelial sodium channel (ENaC) is an important factor in the regulation of sodium absorption, and therefore plays a critical role in minimizing intestinal sodium and water losses. In the present study, we investigated whether IL-13 also acts as a potent modulator of epithelial sodium transport via ENaC, and the signalling components involved. The effect of IL-13 on ENaC was examined in HT-29/B6-GR/MR human colon cells, as well as in mouse distal colon, by measuring amiloride-sensitive short-circuit current (ISC ) in Ussing chambers. The expression levels of ENaC subunits and the cellular components that contribute to ENaC activity were analysed by qRT-PCR and promoter gene assay. We show that IL-13, in both the cell model and in native intestinal tissue, impaired epithelial sodium absorption via ENaC (JNa ) as a result of decreased transcription levels of ß- and γ-ENaC subunits and SGK1, a post-translational regulator of ENaC activity, due to impaired promoter activity. The reduction in JNa was prevented by inhibition of JAK1/2-STAT6 signalling. This inhibition also affected the IL-13-induced decrease in p38 MAPK phosphorylation. The contribution of STAT6 to IL-13-mediated ENaC inactivation was confirmed in a STAT6(-/-) mouse model. In conclusion, these results indicate that IL-13, the levels of which are elevated in ulcerative colitis, contributes to impaired ENaC activity via modulation of the STAT6/p38 MAPK pathways.

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 Crohn's 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.

Oral curcumin ameliorates acute murine campylobacteriosis.

Introduction: Human infections with the food-borne enteropathogen Campylobacter jejuni are responsible for increasing incidences of acute campylobacteriosis cases worldwide. Since antibiotic treatment is usually not indicated and the severity of the enteritis directly correlates with the risk of developing serious autoimmune disease later-on, novel antibiotics-independent intervention strategies with non-toxic compounds to ameliorate and even prevent campylobacteriosis are utmost wanted. Given its known pleiotropic health-promoting properties, curcumin constitutes such a promising candidate molecule. In our actual preclinical placebo-controlled intervention trial, we tested the anti-microbial and anti-inflammatory effects of oral curcumin pretreatment during acute experimental campylobacteriosis. Methods: Therefore, secondary abiotic IL-10-/- mice were challenged with synthetic curcumin via the drinking water starting a week prior oral C. jejuni infection. To assess anti-pathogenic, clinical, immune-modulatory, and functional effects of curcumin prophylaxis, gastrointestinal C. jejuni bacteria were cultured, clinical signs and colonic histopathological changes quantitated, pro-inflammatory immune cell responses determined by in situ immunohistochemistry and intestinal, extra-intestinal and systemic pro-inflammatory mediator measurements, and finally, intestinal epithelial barrier function tested by electrophysiological resistance analysis of colonic ex vivo biopsies in the Ussing chamber. Results and discussion: Whereas placebo counterparts were suffering from severe enterocolitis characterized by wasting symptoms and bloody diarrhea on day 6 post-infection, curcumin pretreated mice, however, were clinically far less compromised and displayed less severe microscopic inflammatory sequelae such as histopathological changes and epithelial cell apoptosis in the colon. In addition, curcumin pretreatment could mitigate pro-inflammatory innate and adaptive immune responses in the intestinal tract and importantly, rescue colonic epithelial barrier integrity upon C. jejuni infection. Remarkably, the disease-mitigating effects of exogenous curcumin was also observed in organs beyond the infected intestines and strikingly, even systemically given basal hepatic, renal, and serum concentrations of pro-inflammatory mediators measured in curcumin pretreated mice on day 6 post-infection. In conclusion, the anti-Campylobacter and disease-mitigating including anti-inflammatory effects upon oral curcumin application observed here highlight the polyphenolic compound as a promising antibiotics-independent option for the prevention from severe acute campylobacteriosis and its potential post-infectious complications.

Myrrh protects against IL-13-induced epithelial barrier breakdown in HT-29/B6 cells.

The oleoresin myrrh has been used for centuries as an anti-inflammatory remedy for a variety of diseases and is said to have a protective effect on the intestinal epithelium. An intact epithelial barrier function is the prerequisite for a healthy gut. Inflammatory and infectious diseases of the intestine, in particular, lead to barrier impairment resulting in leak-flux diarrhea and mucosal immune responses. Therefore, the aim of the present study was to investigate the protective effect of myrrh in an experimental inflammatory situation, namely, under the influence of IL-13, one of the key cytokines in ulcerative colitis. We used human intestinal epithelial HT-29/B6 cell monolayers for functional and molecular assessment of the epithelial barrier under IL-13 and myrrh treatment. IL-13 induced a loss in barrier function that was fully restored with myrrh treatment, as shown by transepithelial electrical resistance measurements. The molecular correlate of the IL-13-mediated barrier dysfunction could be assigned to an upregulation of the channel-forming tight junction (TJ) protein claudin-2 and to a subcellular redistribution of the TJ protein tricellulin, loosening the sealing of tricellular TJs. Moreover, IL-13 exposure leads to an increase in the number of apoptotic cells, contributing to the leak pathway of barrier dysfunction. Myrrh protected against changes in TJ deregulation and decreased the elevated apoptotic ratio under IL-13. The protective effects are mediated through the inhibition of the STAT3 and STAT6 pathway. In conclusion, our results demonstrate that myrrh exhibits antagonizing effects against IL-13-induced barrier impairment in a human intestinal cell model. These data suggest the use of myrrh as a promising option in the treatment of inflammatory bowel disease.

CDT of Clostridioides difficile Induces MLC-Dependent Intestinal Barrier Dysfunction in HT-29/B6 Epithelial Cell Monolayers.

Background: Clostridioides difficile binary toxin (CDT) defines the hypervirulence of strains in nosocomial antibiotic-induced colitis with the highest mortality. The objective of our study was to investigate the impact of CDT on the intestinal epithelial barrier and to enlighten the underlying molecular mechanisms. Methods: Functional measurements of epithelial barrier function by macromolecular permeability and electrophysiology were performed in human intestinal HT-29/B6 cell monolayers. Molecular analysis of the spatial distribution of tight junction protein and cytoskeleton was performed by super-resolution STED microscopy. Results: Sublethal concentrations of CDT-induced barrier dysfunction with decreased TER and increased permeability for 332 Da fluorescein and 4 kDa FITC-dextran. The molecular correlate to the functional barrier defect by CDT was found to be a tight junction protein subcellular redistribution with tricellulin, occludin, and claudin-4 off the tight junction domain. This redistribution was shown to be MLCK-dependent. Conclusions: CDT compromised epithelial barrier function in a human intestinal colonic cell model, even in sublethal concentrations, pointing to barrier dysfunction in the intestine and leak flux induction as a diarrheal mechanism. However, this cannot be attributed to the appearance of apoptosis and necrosis, but rather to an opening of the paracellular leak pathway as the result of epithelial tight junction alterations.

Evaluation of Bile Salts on the Survival and Modulation of Virulence of Aliarcobacter butzleri.

Aliarcobacter butzleri is a Gram-negative bacterium associated with infections of the gastrointestinal tract and widely distributed in various environments. For successful infection, A. butzleri should be able to tolerate various stresses during gastrointestinal passage, such as bile. Bile represents an antimicrobial host barrier that acts against external noxious agents and consists of a variety of bile salts. The intestinal bile salts act as detergents involved in the antimicrobial host defense; although, on the bacterial side, they could also serve as a signal to activate virulence mechanisms. The aim of this work was to understand the effects of bile salts on the survival and virulence of A. butzleri. In our study, A. butzleri was able to survive in the presence of human physiological concentrations of bile salts. Regarding the virulence features, an increase in cellular hydrophobicity, a decrease in motility and expression of flaA gene, as well as an increase in biofilm formation with a concomitant change in the type of biofilm structure were observed in the presence of sub-inhibitory concentration of bile salts. Concerning adhesion and invasion ability, no significant difference was observed. Overall, the results demonstrated that A. butzleri is able to survive in physiological concentrations of bile salts and that exposure to bile salts could change its virulence mechanisms.

Intestinal Barrier in Post-Campylobacter jejuni Irritable Bowel Syndrome.

BACKGROUND: Campylobacter jejuni (C. jejuni) is one of the most common causes of bacterial gastroenteritis worldwide. One sequela of this infection is the development of post-infectious irritable bowel syndrome (PI-IBS). It has been suggested that a dysfunctional intestinal barrier may promote IBS development. We aimed to test this hypothesis against the background of the leaky gut concept for low-grade inflammation in PI-IBS. METHODS: We identified patients with persistent PI-IBS symptoms after C. jejuni infection. During sigmoidoscopy, forceps biopsies were obtained for electrophysiological measurements of epithelial transport and barrier function in miniaturized Ussing devices. C. jejuni absence was checked by PCR and cytokine production with immunohistochemistry. RESULTS: In PI-IBS, the epithelial resistance of the colon epithelium was unaltered, reflecting an intact paracellular pathway. In contrast, temperature-dependent horseradish peroxidase (HRP, 44 kDa) permeation increased. Short-circuit current (Isc) reflecting active anion secretion and ENaC-dependent electrogenic sodium absorption was unaffected. Early endosome antigen-1 (EEA1) and IL-4 levels increased. C. jejuni is not incorporated into the resident microbiota of the colon mucosa in PI-IBS. CONCLUSIONS: In PI-IBS after C. jejuni infection, macromolecule uptake via endocytosis was enhanced, leading to low-grade inflammation with pro-inflammatory cytokine release. The findings will allow C. jejuni-induced pathomechanisms to be targeted during infection and, thereafter to reduce sequelae such as PI-IBS.

A Colonic Organoid Model Challenged with the Large Toxins of Clostridioides difficile TcdA and TcdB Exhibit Deregulated Tight Junction Proteins.

BACKGROUND: Clostridioides difficile toxins TcdA and TcdB are responsible for diarrhea and colitis. Lack of functional studies in organoid models of the gut prompted us to elucidate the toxin's effects on epithelial barrier function and the molecular mechanisms for diarrhea and inflammation. METHODS: Human adult colon organoids were cultured on membrane inserts. Tight junction (TJ) proteins and actin cytoskeleton were analyzed for expression via Western blotting and via confocal laser-scanning microscopy for subcellular localization. RESULTS: Polarized intestinal organoid monolayers were established from stem cell-containing colon organoids to apply toxins from the apical side and to perform functional measurements in the organoid model. The toxins caused a reduction in transepithelial electrical resistance in human colonic organoid monolayers with sublethal concentrations. Concomitantly, we detected increased paracellular permeability fluorescein and FITC-dextran-4000. Human colonic organoid monolayers exposed to the toxins exhibited redistribution of barrier-forming TJ proteins claudin-1, -4 and tricellulin, whereas channel-forming claudin-2 expression was increased. Perijunctional F-actin cytoskeleton organization was affected. CONCLUSIONS: Adult stem cell-derived human colonic organoid monolayers were applicable as a colon infection model for electrophysiological measurements. The TJ changes noted can explain the epithelial barrier dysfunction and diarrhea in patients, as well as increased entry of luminal antigens triggering inflammation.

Impaired Intestinal Permeability of Tricellular Tight Junctions in Patients with Irritable Bowel Syndrome with Mixed Bowel Habits (IBS-M).

BACKGROUND: The underlying pathophysiology of irritable bowel syndrome (IBS) is still unclear. Our aim was to investigate the pathophysiological mechanisms of diarrhea, constipation, and antigen uptake in mixed-type IBS (IBS-M). METHODS: Colonoscopic biopsies were obtained from IBS-M patients. Epithelial transport and barrier function of colonic mucosae were characterized in Ussing chambers using impedance spectroscopy. Mucosal permeability to macromolecules was measured. Western blotting for tight junction (TJ) proteins was performed and their subcellular localization was visualized by confocal microscopy. RNA-sequencing was performed for gene expression and signaling pathway analysis. RESULTS: In IBS-M, epithelial resistance and ENaC-dependent sodium absorption were unchanged, while short-circuit current reflecting chloride secretion was reduced. Concomitantly, epithelial permeability for fluorescein and FITC-dextran-4000 increased. TJ protein expression of occludin decreased, whereas claudins were unaltered. Confocal microscopy revealed the de-localization of tricellulin from tricellular TJs. Involved pathways were detected as proinflammatory cytokine pathways, LPS, PGE2, NGF, and vitamin D. CONCLUSIONS: Decreased anion secretion explains constipation in IBS-M, while ion permeability and sodium absorption were unaltered. Reduced occludin expression resulted in the delocalization of tricellulin from the tricellular TJ, leading to increased macromolecular permeability that contributes to antigen influx into the mucosa and perpetuates a low-grade inflammatory process.

Epithelial Barrier Dysfunction in Diarrhea-Predominant Irritable Bowel Syndrome (IBS-D) via Downregulation of Claudin-1.

BACKGROUND: In patients with diarrhea-predominant irritable bowel syndrome (IBS-D), the diarrheal mechanisms are largely unknown, and they were examined in this study on colon biopsies. METHODS: Electrophysiological measurements were used for monitoring functional changes in the diarrheic colon specimens. In parallel, tight junction protein expression was analyzed by Western blot and confocal laser-scanning microscopy, and signaling pathway analysis was performed using RNA sequencing and bioinformatics. RESULTS: Epithelial resistance was decreased, indicating an epithelial leak flux diarrheal mechanism with a molecular correlate of decreased claudin-1 expression, while induction of active anion secretion and impairment of active sodium absorption via the epithelial sodium channel, ENaC, were not detected. The pathway analysis revealed activation of barrier-affecting cytokines TNF-α, IFN-γ, IL-1ß and IL-4. CONCLUSIONS: Barrier dysfunction as a result of epithelial tight junction changes plays a role in IBS-D as a pathomechanism inducing a leak flux type of diarrhea.

Aerolysin from Aeromonas hydrophila perturbs tight junction integrity and cell lesion repair in intestinal epithelial HT-29/B6 cells.

BACKGROUND: Aeromonads cause a variety of infections, including gastroenteritis, sepsis, and wound necrosis. Pathogenesis of Aeromonas hydrophila and its hemolysin has been characterized, but the mechanism of the epithelial barrier dysfunction is currently poorly understood. METHODS: Human colon epithelial monolayers HT-29/B6 were apically inoculated with clinical isolates of A. hydrophila or with the secreted pore-forming toxin aerolysin. Epithelial resistance and permeability for several markers were determined in Ussing chambers, using 2-path impedance spectroscopy. The subcellular distribution of tight junction (TJ) and cytoskeleton proteins was analyzed by Western blotting and confocal laser-scanning microscopy. RESULTS: A. hydrophila infection induces chloride secretion with a small decrease in transcellular resistance. However, the major effect of A. hydrophila, mediated by its toxin aerolysin, was a sustained reduction of paracellular resistance by retracting sealing TJ proteins from the TJ strands. Aerolysin-treated monolayers showed increased paracellular permeability to FITC-dextran-4000 (0.104 ± 0.014 vs 0.047 ± 0.001 10(-6) cm/s in control; P < .05). Moreover, restitution of epithelial lesions was impaired. The effects were myosin light chain kinase (MLCK) dependent and mediated by intracellular Ca(2+) signaling. CONCLUSIONS: During Aeromonas infection, pore formation by aerolysin impairs epithelial integrity by promoting TJ protein redistribution and consequently affecting wound closure. Thus, Aeromonas-induced diarrhea is mediated by 2 mechanisms, transcellular secretion and paracellular leak flux.