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.

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.

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.

Macromolecule Translocation across the Intestinal Mucosa of HIV-Infected Patients by Transcytosis and through Apoptotic Leaks.

Based on indirect evidence, increased mucosal translocation of gut-derived microbial macromolecules has been proposed as an important pathomechanism in HIV infection. Here, we quantified macromolecule translocation across intestinal mucosa from treatment-naive HIV-infected patients, HIV-infected patients treated by combination antiretroviral therapy, and HIV-negative controls and analyzed the translocation pathways involved. Macromolecule permeability was quantified by FITC-Dextran 4000 (FD4) and horseradish peroxidase (HRP) flux measurements. Translocation pathways were addressed using cold inhibition experiments. Tight junction proteins were characterized by immunoblotting. Epithelial apoptosis was quantified and translocation pathways were further characterized by flux studies in T84 cell monolayers using inducers and inhibitors of apoptosis and endocytosis. In duodenal mucosa of untreated but not treated HIV-infected patients, FD4 and HRP permeabilities were more than a 4-fold increase compared to the HIV-negative controls. Duodenal macromolecule permeability was partially temperature-dependent and associated with epithelial apoptosis without altered expression of the analyzed tight junction proteins. In T84 monolayers, apoptosis induction increased, and both apoptosis and endocytosis inhibitors reduced macromolecule permeability. Using quantitative analysis, we demonstrate the increased macromolecule permeability of the intestinal mucosa in untreated HIV-infected patients. Combining structural and mechanistic studies, we identified two pathways of increased macromolecule translocation in HIV infection: transcytosis and passage through apoptotic leaks.

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.

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.

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.

Clinical Significance of Claudin Expression in Oral Squamous Cell Carcinoma.

A change in claudin expression has been demonstrated in various tumors. The present study specifically compares claudin expression in oral squamous cell carcinoma (OSCC) with healthy oral epithelium from the same individual and analyzes the association between claudin expression and the clinically relevant course parameters. Our study includes tissue samples and clinically relevant follow-up data from 60 patients with primary and untreated OSCC. The oral mucosa was analyzed via Western blot for the expression of claudin-1, -2, -3, -4, -5, and -7. Importantly, the tumor and healthy tissues were obtained pairwise from patients, allowing for intraindividual comparisons. Both the healthy and tumor epithelium from the oral cavity did not express the claudin-3 protein. The intraindividual comparison revealed that, in OSCC, claudin-2 expression was higher, and the expression of claudin-4, -5, and -7 was lower than in healthy epithelium. An association was found between increased claudin-2 expression and shorter relapse-free survival. In addition, the reduced expression of claudin-4 had a negative impact on relapse-free survival. Furthermore, associations between the reduced expression of claudin-7 and the stage of a tumor, or the presence of lymph node metastases, were found. Thus, the expression level of claudin-2, -4, and -7 appears to be predictive of the diagnosis and prognosis of OSCC.

Unraveling the intestinal epithelial barrier in cyanotoxin microcystin-treated Caco-2 cell monolayers.

Microcystin is a widespread cyanobacterial toxin that affects the intestine to produce diarrheal symptoms after ingestion of freshwater blue-green algae. Our study aimed to characterize the mechanism by which the toxin leads to diarrhea via epithelial barrier dysfunction in a small intestine Caco-2 cell model. Microcystin-treated human Caco-2 epithelial monolayers were functionally and molecularly analyzed for barrier dysfunction. Tight junctions (TJs) and cell damage were analyzed in relation to transepithelial electrical resistance (TER) changes. TER of microcystin-treated Caco-2 cells was reduced by 65% of the initial value after 24 h; concomitantly, permeability for fluorescein increased 2.6-fold. Western blot analysis showed reduced claudin-1 expression, while expression of claudin-3 and -4 remained unchanged. Super-resolution stimulated emission depletion microscopy revealed that TJ integrity was compromised by fraying and splitting of the TJ domain of the epithelial cells. Epithelial apoptosis did not significantly contribute to epithelial barrier dysfunction, while cytoskeletal actomyosin constriction was associated with TJ disintegration and the barrier defect. Our results indicate that microcystin causes intestinal barrier leakiness, which helps to explain the leak flux type of diarrhea as the main pathomechanism after ingestion of cyanobacterial toxin.

Escherichia coli Alpha-Hemolysin HlyA Induces Host Cell Polarity Changes, Epithelial Barrier Dysfunction and Cell Detachment in Human Colon Carcinoma Caco-2 Cell Model via PTEN-Dependent Dysregulation of Cell Junctions.

Escherichia coli (E. coli) of the B2 phylotype reside in human and animal intestines. The bacteria possess pathogenicity factors such as α-hemolysin (HlyA) that can induce intestinal epithelial leaks. We addressed the questions which host cell processes were dysregulated by E. coli HlyA that can potentiate intestinal diseases. The colon carcinoma cell line Caco-2 was infected by HlyA+ E. coli. Cell polarity regulation was analyzed by live cell imaging for the phosphatidylinositol-4,5-bisphosphate (PIP2) abundance. In Caco-2 monolayers, transepithelial electrical resistance was measured for characterization of barrier function. Cell proliferation and separation were assessed microscopically. Epithelial regulation and cell signaling were analyzed by RNA-Seq and Ingenuity Pathway Analysis (IPA). Our main findings from E. coli HlyA toxinogenicity in the colon carcinoma cell line are that (i) PIP2 at the membrane decrease, (ii) PTEN (phosphatase and tensin homolog) inhibition leads to cell polarity changes, (iii) epithelial leakiness follows these polarity changes by disruption of cell junctions and (iv) epithelial cell detachment increases. HlyA affected pathways, e.g., the PTEN and metastasis signaling, were identified by RNA-Seq bioinformatics calculations in IPA. In conclusion, HlyA affects cell polarity, thereby inducing epithelial barrier dysfunction due to defective tight junctions and focal leak induction as an exemplary mechanism for leaky gut.

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.

Expression of tricellular tight junction proteins and the paracellular macromolecule barrier are recovered in remission of ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) has a relapsing and remitting pattern, wherein the underlying mechanisms of the relapse might involve an enhanced uptake of luminal antigens which stimulate the immune response. The tricellular tight junction protein, tricellulin, takes charge of preventing paracellular passage of macromolecules. It is characterized by downregulated expression in active UC and its correct localization is regulated by angulins. We thus analyzed the tricellulin and angulin expression as well as intestinal barrier function and aimed to determine the role of tricellulin in the mechanisms of relapse. METHODS: Colon biopsies were collected from controls and UC patients who underwent colonoscopy at the central endoscopy department of Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin. Remission of UC was defined basing on the clinical appearance and a normal Mayo endoscopic subscore. Intestinal barrier function was evaluated by electrophysiological and paracellular flux measurements on biopsies mounted in Ussing chambers. RESULTS: The downregulated tricellulin expression in active UC was recovered in remission UC to control values. Likewise, angulins were in remission UC at the same levels as in controls. Also, the epithelial resistance which was decreased in active UC was restored in remission to the same range as in controls, along with the unaltered paracellular permeabilities for fluorescein and FITC-dextran 4 kDa. CONCLUSIONS: In remission of UC, tricellulin expression level as well as intestinal barrier functions were restored to normal, after they were impaired in active UC. This points toward a re-sealing of the impaired tricellular paracellular pathway and abated uptake of antigens to normal rates in remission of UC.

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).

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.

Resveratrol Alleviates Acute Campylobacter jejuni Induced Enterocolitis in a Preclinical Murine Intervention Study.

The polyphenolic compound resveratrol has been shown to exert health-beneficial properties. Given globally emerging Campylobacter infections in humans, we addressed potential anti-pathogenic, immuno-modulatory and intestinal epithelial barrier preserving properties of synthetic resveratrol in the present preclinical intervention study applying a murine acute campylobacteriosis model. Two days following peroral C. jejuni infection, secondary abiotic IL-10-/- mice were either subjected to resveratrol or placebo via the drinking water. Whereas placebo mice suffered from acute enterocolitis at day 6 post-infection, resveratrol treatment did not only lead to improved clinical conditions, but also to less pronounced colonic epithelial apoptosis as compared to placebo application. Furthermore, C. jejuni induced innate and adaptive immune cell responses were dampened in the large intestines upon resveratrol challenge and accompanied by less colonic nitric oxide secretion in the resveratrol versus the placebo cohort. Functional analyses revealed that resveratrol treatment could effectively rescue colonic epithelial barrier function in C. jejuni infected mice. Strikingly, the disease-alleviating effects of resveratrol could additionally be found in extra-intestinal and also systemic compartments at day 6 post-infection. For the first time, our current preclinical intervention study provides evidence that peroral resveratrol treatment exerts potent disease-alleviating effects during acute experimental campylobacteriosis.

Zinc prevents intestinal epithelial barrier dysfunction induced by alpha-hemolysin-producing Escherichia coli 536 infection in porcine colon.

Zinc treatment is beneficial for infectious diarrhea or colitis. This study aims to characterize the pathomechanisms of the epithelial barrier dysfunction caused by alpha-hemolysin (HlyA)-expressing Escherichia coli in the colon mucosa and the mitigating effects of zinc ions. We performed Ussing chamber experiments on porcine colon epithelium and infected the tissues with HlyA-producing E. coli. Colon mucosa from piglets was obtained from a feeding trial with defined normal or high dose zinc feeding (pre-conditioning). Additional to the zinc feeding, zinc was added to the luminal compartment of the Ussing chamber. Transepithelial electrical resistance (TER) was measured during the infection of the living tissue and subsequently the tissues were immuno-stained for confocal microscopy. Zinc applied to the luminal compartment was effective in preventing from E. coli-induced epithelial barrier dysfunction in Ussing chamber experiments. In contrast, zinc pre-conditioning of colon mucosae when zinc ions were missing subsequently in the luminal compartment was not sufficient to prevent epithelial barrier impairment during E. coli infection. The pathological changes caused by E. coli HlyA were alterations of tight junction proteins claudin-4 and claudin-5, focal leak formation, and cell exfoliation which reflected the paracellular barrier defect measured by a reduced TER. In microscopic analysis of luminal zinc-treated mucosae these changes were absent. In conclusion, continuous presence of unbound zinc ions in the luminal compartment is essential for the protective action of zinc against E. coli HlyA. This suggests the usage of zinc as therapeutic regimen, while prophylactic intervention by high dietary zinc loads may be less useful.

Altered Structural Expression and Enzymatic Activity Parameters in Quiescent Ulcerative Colitis: Are These Potential Normalization Criteria?

Mucosal healing determined by endoscopy is currently the remission standard for ulcerative colitis (UC). However, new criteria for remission are emerging, such as histologic normalization, which appears to correlate better to the risk of relapse. Here, we study mucosal healing on a molecular and functional level in quiescent UC. We obtained endoscopic biopsies from 33 quiescent UC patients and from 17 controls. Histology was assessed using Geboes score. Protein and mRNA levels were evaluated for the tight junction proteins claudin-2, claudin-4, occludin, and tricellulin, as well as Cl-/HCO3- exchanger DRA, and cyclo-oxygenase enzymes (COX-1, COX-2). The mucosal activity of COX-1 and COX-2 enzymes was assessed in modified Ussing chambers, measuring electrogenic ion transport (short-circuit current, SCC). Chronic inflammation was present in most UC patients. The protein level of claudin-4 was reduced, while mRNA-levels of claudin-2 and claudin-4 were upregulated in UC patients. Surprisingly, the mRNA level of COX-1 was downregulated, but was unaltered for COX-2. Basal ion transport was not affected, while COX-2 inhibition induced a two-fold larger decrease in SCC in UC patients. Despite being in clinical and endoscopic remission, quiescent UC patients demonstrated abnormal mucosal barrier properties at the molecular and functional level. Further exploration of mucosal molecular signature for revision of current remission standards should be considered.

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.