GAL3ST1 Deficiency Reduces Epithelial-Mesenchymal Transition and Tumorigenic Capacity in a Cholangiocarcinoma Cell Line.

Cholangiocarcinoma (CCA), or bile duct cancer, is the second most common liver malignancy, with an increasing incidence in Western countries. The lack of effective treatments associated with the absence of early symptoms highlights the need to search for new therapeutic targets for CCA. Sulfatides (STs), a type of sulfoglycosphingolipids, have been found in the biliary tract, with increased levels in CCA and other types of cancer. STs are involved in protein trafficking and cell adhesion as part of the lipid rafts of the plasma membrane. We aimed to study the role of STs in CCA by the genetic targeting of GAL3ST1, an enzyme involved in ST synthesis. We used the CRISPR-Cas9 system to generate GAL3ST1-deficient TFK1 cells. GAL3ST1 KO cells showed lower proliferation and clonogenic activity and reduced glycolytic activity compared to TFK1 cells. Polarized TFK1 GAL3ST1 KO cells displayed increased transepithelial resistance and reduced permeability compared to TFK1 wt cells. The loss of GAL3ST1 showed a negative effect on growth in 30 out of 34 biliary tract cancer cell lines from the DepMap database. GAL3ST1 deficiency partially restored epithelial identity and barrier function and reduced proliferative activity in CCA cells. Sulfatide synthesis may provide a novel therapeutic target for CCA.

The effects of citrus flavonoids and their metabolites on immune-mediated intestinal barrier disruption using an in vitro co-culture model.

Hesperidin and naringin are citrus flavonoids with known anti-oxidative and anti-inflammatory properties. Evidence from previous studies indicates that both these compounds and the metabolites that are formed during intestinal metabolism are able to exert beneficial effects on intestinal barrier function and inflammation. However, so far, studies investigating the relative contributions of the various compounds are lacking. Therefore, we assessed the effect of citrus flavonoids and their intestinal metabolites on immune-mediated barrier disruption in an in vitro co-culture model. Caco-2 cell monolayers were placed in co-culture with phorbol 12-myristate 13-acetate-stimulated THP-1-Blue™ NF-κB cells for 30 h. At baseline, the citrus flavonoids and their metabolites were added to the apical compartment (50 or 100 µM per compound). After 24 h, THP-1 cells were incubated with lipopolysaccharide (LPS) in the basolateral compartment for 6 h. Incubation with citrus flavonoids and their metabolites did not induce changes in transepithelial electrical resistance, fluorescein isothiocyanate-dextran 4 kDa permeation or gene expression of barrier-related genes for any of the compounds tested. After LPS stimulation, NF-κB activity was significantly inhibited by all compounds (100 µM) except for one metabolite (all P ≤ 0·03). LPS-induced production of the cytokines IL-8, TNF-α and IL-6 was inhibited by most compounds (all P < 0·05). However, levels of IL-1ß were increased, which may contribute to the lack of an improved barrier effect. Overall, these results suggest that citrus flavonoids may decrease intestinal inflammation via reduction of NF-κB activity and that the parent compounds and their metabolites formed during intestinal metabolism are able to exert comparable effects.

Interleukin-28A induces epithelial barrier dysfunction in CD patient-derived intestinal organoids.

Intestinal barrier dysfunction is a pathogenic hallmark in Crohn's disease (CD). Identifying key players that regulate intestinal barrier may provide novel leads for therapeutic intervention. Interleukin-28A (IL-28A) is a newly identified IL-10/interferon cytokine family member, with its most implicated function being antiviral and anti-proliferative properties. However, the role and underlying mechanisms of IL-28A in the regulation of epithelial barrier in CD remain so far unexplored. IL-28A levels were measured in the plasma and biopsies of CD patients and healthy subjects. CD patient-derived intestinal organoids were characterized by differentiation gene markers and then exposed to TNF-α, IFN-γ, IL-1ß or LPS, or IL-28A with or without GLPG0634 (filgotinib). Epithelial permeability was assessed by FITC-D4 flux. Expression of junctional components was analyzed by qRT-PCR, immunofluorescence staining, or Western blotting. JAK-STAT activity was analyzed by Western blotting. IL-28A levels were significantly increased in the plasma and biopsies from active patients with CD as compared with healthy subjects. IL-28A and its receptor complex IL-28AR/IL-10R2 were detected in CD patient-derived intestinal organoids and showed a selective response to IFN-γ exposure. IL-28A triggered epithelial barrier disruption and accompanied by reduced ZO-1 and E-cadherin expression. This effect was mediated by JAK-STAT1 pathway. Pre-incubation with the JAK1 inhibitor filgotinib ameliorated the barrier dysfunction induced by IL-28A. These results identified IL-28A as a novel regulator of epithelial barrier function and could be a putative target for CD treatment. We provide novel basic evidence that restoring intestinal barrier is a potential mechanism that contributes to the clinical benefits of JAK1 inhibitor in patients with CD.NEW & NOTEWORTHY IL-28A levels were significantly increased in the plasma and biopsies from active patients with CD as compared with healthy subjects. IFN-γ exposure stimulated IL-28A expression in intestinal organoids. Partially mimicking the effect of IFN-γ, IL-28A impaired epithelial barrier function and disrupted junctional components through the activation of JAK-STAT1 signaling, whereas JAK1 inhibitor ameliorated the above-mentioned effects of IL-28A. These findings highlight the newly identified cytokine IL-28A as a novel contributor to CD pathogenesis and could be a putative target for CD treatment. We also provide new evidence for potential applications of JAK inhibition in CD therapy.

The Role of Brown Adipose Tissue in the Development and Treatment of Nonalcoholic Steatohepatitis: An Exploratory Gene Expression Study in Mice.

Brown adipose tissue (BAT) might be a beneficial mediator in the development and treatment of nonalcoholic steatohepatitis (NASH). We aim to evaluate the gene expression of BAT activity-related genes during the development and the dietary and surgical treatment of NASH. BAT was collected from male C57BL/6J mice that received a high fat-high sucrose diet (HF-HSD) or a normal chow diet (NCD) for 4 and 20 weeks (n=8-9 per dietary group and timepoint) and from mice that underwent dietary intervention (return to NCD) (n=8), roux-en-y gastric bypass (RYGB) (n=6), or sham procedure (n=6) after 12 weeks HF-HSD. Expression of BAT genes involved in lipid metabolism (Cd36 and Cpt1b; p<0.05) and energy expenditure (Ucp1 and Ucp3; p<0.05) were significantly increased after 4 weeks HF-HSD compared with NCD, whereas in the occurrence of NASH after 20 weeks HF-HSD no difference was observed. We observed no differences in gene expression regarding lipid metabolism or energy expenditure at 8 weeks after dietary intervention (no NASH) compared with HF-HSD mice (NASH), nor in mice that underwent RYGB compared with SHAM. However, dietary intervention and RYGB both decreased the BAT gene expression of inflammatory cytokines (Il1b, Tnf-α and MCP-1; p<0.05). Gene expression of the batokine neuregulin 4 was significantly decreased after 20 weeks HF-HSD (p<0.05) compared with NCD, but was restored by dietary intervention and RYGB (p<0.05). In conclusion, BAT is hallmarked by dynamic alterations in the gene expression profile during the development of NASH and can be modulated by dietary intervention and bariatric surgery.

Exploration of the Esophageal Mucosal Barrier in Non-Erosive Reflux Disease.

In the absence of visible mucosal damage, it is hypothesized that the esophageal mucosal barrier is functionally impaired in patients with non-erosive reflux disease (NERD). The aim of the present study was to perform an exploratory analysis of the mucosal barrier in NERD compared to erosive esophagitis (EE) and controls. A second aim was to explore TRPV1 gene transcription in relation to the mucosal barrier function and heartburn symptoms. In this prospective study, 10 NERD patients, 11 patients with active erosive esophagitis and 10 healthy volunteers were included. Biopsies from non-eroded mucosa were obtained for (1) ex vivo analyses (Ussing chamber) of transepithelial electrical resistance (TEER) and permeability (2) gene transcription of tight-junction proteins and transient receptor potential vanilloid subfamily member 1 (TRPV1). No differences in TEER or permeability were found between NERD and healthy volunteers, whereas TEER was lower in patients with erosive esophagitis. TRPV1 gene transcription was not significantly different between EE, NERD and controls. CONCLUSIONS: esophageal mucosal barrier function and TRPV1 transcription is not significantly altered in NERD patients. Future research is needed to explore other potential mechanisms that may account for the high symptom burden in these patients.

Is intestinal oxidative stress involved in patients with compensated liver cirrhosis?

BACKGROUND: Liver cirrhosis is associated with intestinal epithelial barrier dysfunction, which may be affected by oxidative stress. Studies in cirrhotic rats provided evidence for intestinal oxidative stress, but studies in cirrhotic patients are scarce. We have shown intestinal barrier dysfunction in patients with compensated cirrhosis. AIM: The present study aimed to investigate whether oxidative stress occurs in the intestinal mucosa of compensated cirrhotic patients and may contribute to barrier dysfunction. MATERIAL AND METHODS: Oxidative stress was studied in duodenal and sigmoid biopsies from 15 cirrhotic patients and 22 controls by analyzing transcription of genes involved in glutathione and uric acid metabolism using quantitative real-time polymerase chain reaction. Protein levels of glutathione and glutathione disulphide were measured and the glutathione/glutathione disulphide ratio was calculated as marker of oxidative stress. In addition, intestinal myeloperoxidase and fecal calprotectin were determined. RESULTS: Gene transcription of glutathione synthetase and glutathione reductase were significantly different in duodenal and sigmoid biopsies of cirrhotic patients vs. controls, but no alterations were found for other genes nor for glutathione, glutathione disulphide, glutathione/glutathione disulphide ratio and intestinal myeloperoxidase and fecal calprotectin concentrations. CONCLUSION: This study did not find indications for oxidative stress and low-grade inflammation in the small and large intestine of stable compensated cirrhotic patients. Although these preliminary findings need further validation, we found intestinal oxidative stress not to be a major mechanism contributing to epithelial barrier dysfunction in patients with compensated cirrhosis.

Corticosteroid enhances epithelial barrier function in intestinal organoids derived from patients with Crohn's disease.

Corticosteroids (CS), first-line therapeutics for Crohn's disease (CD) with moderate or severe disease activity, were found to restore intestinal permeability in CD patients, whereas the underlying molecular events are still largely unknown. This study aimed to investigate the effect and mechanisms of CS prednisolone on epithelial barrier using CD patient-derived intestinal organoids. 3D intestinal organoids were generated from colon biopsies of inactive CD patients. To mimic the inflammatory microenvironment, a mixture of cytokines containing TNF-α, IFN-γ, and IL-1ß were added to the organoid culture with or without pre-incubation of prednisolone or mifepristone. Epithelial permeability of the organoids was assessed by FITC-D4 flux from the basal to luminal compartment using confocal microscopy. Expression of junctional components were analyzed by qRT-PCR, immunofluorescence staining, and western blot. Activity of signaling pathways were analyzed using western blot. Exposure of the cytokines significantly disrupted epithelial barrier of the intestinal organoids, which was partially restored by prednisolone. On the molecular level, the cytokine mixture resulted in a significant reduction in E-cadherin and ILDR-1, an increase in CLDN-2, MLCK, and STAT1 phosphorylation, whereas prednisolone ameliorated the abovementioned effects induced by the cytokine mixture. This study demonstrates that prednisolone confers a direct effect in tightening the epithelial barrier, identifies novel junctional targets regulated by prednisolone, and underscores intestinal barrier restoration as a potential mechanism that contributes to the clinical efficacy of prednisolone in CD patients. KEY MESSAGES: Prednisolone confers a direct preventive effect against cytokine-induced barrier dysfunction. Prednisolone regulates the expression of CLDN-2, E-cadherin, and ILDR-1. The effect of prednisolone is GR-, MLCK-, and STAT1-dependent.

Galacto-oligosaccharides supplementation in prefrail older and healthy adults increased faecal bifidobacteria, but did not impact immune function and oxidative stress.

BACKGROUND & AIMS: Ageing is associated with an increased risk of frailty, intestinal microbiota perturbations, immunosenescence and oxidative stress. Prebiotics such as galacto-oligosaccharides (GOS) may ameliorate these ageing-related alterations. We aimed to compare the faecal microbiota composition, metabolite production, immune and oxidative stress markers in prefrail elderly and younger adults, and investigate the effects of GOS supplementation in both groups. METHODS: In a randomised controlled cross-over study, 20 prefrail elderly and 24 healthy adults received 21.6 g/day Biotis™ GOS (containing 15.0 g/day GOS) or placebo. Faecal 16S rRNA gene-based microbiota and short-chain fatty acids were analysed at 0, 1 and 4 weeks of intervention.Volatile organic compounds were analysed in breath, and stimulated cytokine production, CRP, malondialdehyde, trolox equivalent antioxidant capacity (TEAC) and uric acid (UA) in blood at 0 and 4 weeks. RESULTS: Principle coordinate analysis showed differences in microbial composition between elderly and adults (P≤0.05), with elderly having lower bifidobacteria (P≤0.033) at baseline. In both groups, GOS affected microbiota composition (P≤0.05), accompanied by increases in bifidobacteria (P<0.001) and decreased microbial diversity (P≤0.023). Faecal and breath metabolites, immune and oxidative stress markers neither differed between groups (P ≥ 0.125) nor were affected by GOS (P ≥ 0.236). TEAC values corrected for UA were higher in elderly versus adults (P<0.001), but not different between interventions (P ≥ 0.455). CONCLUSIONS: Elderly showed lower faecal bifidobacterial (relative) abundance than adults, which increased after GOS intake in both groups. Faecal and breath metabolites, parameters of immune function and oxidative stress were not different at baseline, and not impacted by GOS supplementation. CLINICALTRIALS. GOV WITH STUDY ID NUMBER: NCT03077529.

Intestinal barrier function is maintained with aging - a comprehensive study in healthy subjects and irritable bowel syndrome patients.

Animal studies have shown that intestinal barrier function is compromised with aging. We aimed to assess the effects of aging on intestinal barrier function in humans in vivo and ex vivo. In this cross-sectional study, healthy subjects and subjects with irritable bowel syndrome (IBS) of older (65-75 years) and young adult age (18-40 years) were compared. In vivo gastrointestinal site-specific permeability was assessed by a multi-sugar test, taking into account potential confounders. Sigmoid biopsies were collected from subgroups of healthy young adults and elderly for ex vivo Ussing chamber experiments, gene transcription of barrier-related genes and staining of junctional proteins. No significant differences between healthy young adults and elderly were found for small intestinal, colonic and whole gut permeability (P ≥ 0.142). In IBS patients, gastroduodenal and colonic permeability did not differ significantly (P ≥ 0.400), but small intestinal and whole gut permeability were higher in elderly versus young adults (P ≤ 0.009), mainly driven by the IBS-diarrhea subtype. Ussing chamber experiments with or without stressor (P ≥ 0.052), and relative expression of intestinal barrier-related genes (P ≥ 0.264) showed no significant differences between healthy elderly and young adults, as confirmed by immunofluorescent stainings. Overall, the functional capacity of the intestinal barrier is maintained in elderly.

The Impact of Pectin Supplementation on Intestinal Barrier Function in Healthy Young Adults and Healthy Elderly.

Intestinal barrier function is suggested to decrease with aging and may be improved by pectin intake. The aim of this study was to investigate the effects of four weeks pectin supplementation on gastrointestinal barrier function in vivo and ex vivo in different age groups. In a randomized, double-blind, placebo-controlled, parallel study, 52 healthy young adults (18-40 years) and 48 healthy elderly (65-75 years) received 15 g/day pectin or placebo for four weeks. Pre- and post-intervention, in vivo gastrointestinal permeability by a multisugar test, and defense capacity in mucosal samples were assessed. Sigmoid biopsies were collected post-intervention from subgroups for Ussing chamber experiments and gene transcription of barrier-related genes. Pectin intervention did not affect in vivo gastroduodenal, small intestinal, colonic, and whole gut permeability in young adults nor in elderly (p ≥ 0.130). Salivary and fecal sIgA and serum IgA were not significantly different between pectin versus placebo in both age groups (p ≥ 0.128). In both young adults and elderly, no differences in transepithelial electrical resistance and fluorescein flux (p ≥ 0.164) and relative expression of genes analyzed (p ≥ 0.222) were found between pectin versus placebo. In conclusion, intestinal barrier function was not affected by four weeks pectin supplementation neither in healthy young adults nor in healthy elderly.

Modulation of Intestinal Epithelial Permeability by Plasma from Patients with Crohn's Disease in a Three-dimensional Cell Culture Model.

Intestinal epithelial barrier is affected by multiple factors, such as tumour necrosis factor-α (TNF-α). Plasma concentration of TNF-α is higher in patients with Crohn's disease (CD) than healthy controls (HC) and correlates positively with disease activity. This study aimed to determine the effect of plasma from active, inactive CD patients on intestinal barrier function and to investigate the underlying mechanism. Plasma samples were collected from CD patients and HC. 3D Caco-2 cysts were treated with plasma or TNF-α, with or without pre-incubation of adalimumab (a monoclonal antibody that antagonizes TNF-α) or JNK inhibitor SP600125. The results demonstrated that exposure of the cysts to plasma from CD patients resulted in enhanced paracellular permeability in a disease activity-dependent manner. Compared to HC, active CD plasma decreased ZO-1 and OCCLUDIN expression on mRNA and protein levels, and led to an increased JNK phosphorylation. Pre-incubation with adalimumab or SP600125 ameliorated TJ disruption and barrier dysfunction induced by plasma from CD patients. These results indicate that plasma from CD patients is able to induce epithelial barrier disruption, in part through TNF-α induced TJs modulation. The data also demonstrate an involvement of MAPK pathway, in particular the JNK isoform, in CD patient plasma-induced barrier dysfunction.