Graphical Abstract Abstract Objective: Intestinal inflammation is linked to gut dysbiosis and disrupted host–microbe interactions. This study investigated four plant extracts rich in polyphenol – pomegranate fruit peel, lemon balm leaves, cinnamon bark, and grape pomace – for their potential prebiotic activities and protective effects in a mouse model of mild intestinal inflammation.Methods: Mice received extracts orally for 4 weeks prior to colitis induction with dextran sulfate sodium.Results: All four extracts reduced colonic inflammation markers, and only grape pomace delayed disease onset. Extracts modified gut microbiota composition during colitis, differentially influencing the abundance of beneficial taxa, pathobionts, hydrogen sulfide producers, and mucus-associated species, such as Akkermansia muciniphila. Metabolomic analysis revealed extract-specific alterations in fecal metabolite profiles, particularly regarding levels of ethanol, glycine and amino acids. Colonic gene transcript profiling showed that grape pomace had the most beneficial effects on the expression of genes involved in epithelial barrier function, microbial sensing, inflammation signaling and innate immunity during colitis. Histological and imaging data further confirmed that grape pomace preserved tight-junction integrity and limited bacterial translocation during colitis.Conclusion: The findings demonstrate that the four polyphenol-rich extracts prevent colitis-induced intestinal damage, likely through modulation of gut microbiota, fecal metabolites and barrier function. Of them, grape pomace extract induced the most promising effect against colitis, combining improved clinical symptoms and restoration of host–microbiome homeostasis.

Thrombin is a serine protease produced by the intestinal epithelium that contributes to mucosal homeostasis by limiting microbial encroachment. In Crohn's disease, mucosal thrombin activity is markedly increased, suggesting a role in disease pathophysiology. We hypothesized that excessive luminal thrombin disrupts host-microbiota interactions and promotes a pathogenic microbial phenotype. Thrombin levels were significantly elevated in fecal samples from a subset of Crohn's disease patients compared with healthy controls. Ex vivo experiments using human colonic biopsy-derived microbiota, cultured as polymicrobial biofilms, revealed that thrombin exposure disrupted biofilm integrity, enriched the protein-rich extracellular matrix, and promoted bacterial dispersal. Dispersed bacteria displayed enhanced adhesion to intestinal epithelial cells and triggered inflammatory and antimicrobial responses through Toll-Like Receptor 5 signaling. Although the global taxonomic composition showed limited changes, metatranscriptomic analyses demonstrated distinct microbial activity profiles in the presence of thrombin. In vivo, intracolonic thrombin altered mucosal biofilm organization in mice, and the transfer of thrombin-exposed mucosal microbiota to germ-free recipients induced mucosal inflammation and bacterial translocation. Finally, in a rat model of colitis, pharmacological inhibition of thrombin activity restored mucosal biofilm architecture and reduced tissue damage. These findings identify thrombin as a host-associated modulator of microbial functional dysbiosis in patients with Crohn's disease. By driving a shift toward a pathogenic microbial phenotype, excessive thrombin links epithelial activity with altered host-microbiota interactions and mucosal inflammation. Thrombin measurement in fecal samples may represent a non-invasive marker of microbiota pathogenicity, and therapeutic targeting of thrombin offers a promising complementary strategy to immune-directed treatments in Crohn's disease.

BACKGROUND:Postoperative Ileus (POI) is an iatrogenic complication characterized by a temporary paralysis of gastrointestinal transit, leading to food intolerance, nausea, vomiting, and thus prolonged hospitalization. The severity of POI is influenced by surgical trauma, particularly in intestinal surgeries, which have a high complication rate. To date, no animal model has precisely replicated POI in the context of digestive sutures or anastomoses, procedures common in human digestive resections. METHODS:To induce POI, mice underwent different surgeries. The surgical procedure involved a midline laparotomy, externalization of the small intestine following external manipulation through moistened cotton applicators, mimicking the surgeon's action when searching for an intestinal lesion. An ileo-ileal anastomosis was also performed to ensure relevance to human surgical intervention. Intestinal transit was measured by assessing gastric emptying, gastrointestinal transit time (gavage with charcoal, fecal output), and gut motility (isotonic contraction). Postoperative inflammation is assessed in different tissue layers and areas of theintestine. KEY RESULTS:The externalization of the small intestine with caecum and manipulation for 10 min induced a pathological postoperative ileus. This simple gesture induced a decrease in intestinal transit comparable to the surgical intervention, ileo-ileal anastomosis. The model showed decreased gastric emptying and reduced ileal muscle contraction, accompanied by neutrophil and monocyte/macrophage infiltration in the external muscularis. CONCLUSIONS AND INFERENCES:The developed procedure enables inducing postoperative ileus in mice in a very simple and reproducible way that does not require any specific equipment, mimics clinical practice, and reproduces traits of human pathology.

Background and Aims Thrombin levels in the colon of Crohn’s disease patients have recently been found to be elevated 100-fold compared with healthy controls. Our aim was to determine whether and how dysregulated thrombin activity could contribute to local tissue malfunctions associated with Crohn’s disease. Methods Thrombin activity was studied in tissues from Crohn’s disease patients and healthy controls. Intracolonic administration of thrombin to wild-type or protease-activated receptor-deficient mice was used to assess the effects and mechanisms of local thrombin upregulation. Colitis was induced in rats and mice by the intracolonic administration of trinitrobenzene sulphonic acid. Results Active forms of thrombin were increased in Crohn’s disease patient tissues. Elevated thrombin expression and activity were associated with intestinal epithelial cells. Increased thrombin activity and expression were also a feature of experimental colitis in rats. Colonic exposure to doses of active thrombin comparable to what is found in inflammatory bowel disease tissues caused mucosal damage and tissue dysfunctions in mice, through a mechanism involving both protease-activated receptors -1 and -4. Intracolonic administration of the thrombin inhibitor dabigatran, as well as inhibition of protease-activated receptor-1, prevented trinitrobenzene sulphonic acid-induced colitis in rodent models. Conclusions Our data demonstrated that increased local thrombin activity, as it occurs in the colon of patients with inflammatory bowel disease, causes mucosal damage and inflammation. Colonic thrombin and protease-activated receptor-1 appear as possible mechanisms involved in mucosal damage and loss of function and therefore represent potential therapeutic targets for treating inflammatory bowel disease.

Imbalance between proteases and their inhibitors plays a crucial role in the development of Inflammatory Bowel Diseases (IBD). Increased elastolytic activity is observed in the colon of patients suffering from IBD. Here, we aimed at identifying the players involved in elastolytic hyperactivity associated with IBD and their contribution to the disease. We revealed that epithelial cells are a major source of elastolytic activity in healthy human colonic tissues and this activity is greatly increased in IBD patients, both in diseased and distant sites of inflammation. This study identified a previously unrevealed production of elastase 2A (ELA2A) by colonic epithelial cells, which was enhanced in IBD patients. We demonstrated that ELA2A hyperactivity is sufficient to lead to a leaky epithelial barrier. Epithelial ELA2A hyperactivity also modified the cytokine gene expression profile with an increase of pro-inflammatory cytokine transcripts, while reducing the expression of pro-resolving and repair factor genes. ELA2A thus appears as a novel actor produced by intestinal epithelial cells, which can drive inflammation and loss of barrier function, two essentials pathophysiological hallmarks of IBD. Targeting ELA2A hyperactivity should thus be considered as a potential target for IBD treatment.

BackgroundCurrent therapies for Inflammatory Bowel Disease (IBD) are unsatisfactory for proper tissue healing. Serine proteases belong to locally produced host factors that can fuel inflammatory processes in tissue from IBD patients, in part through activation of Protease‐Activated Receptors (PAR). We have recently discovered that intestinal epithelium was able to produce active thrombin, suggesting that mucosa itself could be an important source of high thrombin in IBD.ObjectivesWe first aimed to determine whether mucosal thrombin was upregulated in animal models of colitis and in tissues from IBD patients. We then determined whether local thrombin upregulation could contribute to local tissue malfunctions. Finally, we evaluated therapeutic feasibility of local delivering of either direct thrombin inhibitors or PAR antagonist in animal models of colitis.MethodsColonic tissue samples were obtained from diagnosed IBD patients undergoing colonoscopy at the Toulouse Hospital. Colitis was induced by administering trinitrobenzene sulfonic acid (TNBS) in the colon of Wistar rats or C57Bl6 mice. Human tissue collection and animal procedures received ethical approval from local ethic committees. Thrombin (100 U/ml, 10 days), direct thrombin inhibitor (dabigatran, 1 μg/kg, 4 days) and PAR1 antagonist (Vorapaxar, 2.5 mg/kg, 7 days) were administered in the colon of healthy or TNBS animals under light anesthesia. At time of the sacrifice, colonic tissues were harvested and disease severity was assessed. Thrombin expression was detected using PCR, western blot and immunofluorescence. Thrombin activity was quantified in tissue supernatants using specific enzymatic assays.ResultsWe confirmed an increased thrombin protein expression in human mucosal tissue by immunofluorescence and western blots. We found that some, but not all, forms of active thrombin were upregulated, particularly in tissues from Crohn’s disease patients. As observed in human, we found that increased thrombin mRNA expression and activity is also a feature of colitis in animal models of colitis. We demonstrated in vivo that colonic exposure to high dose of active thrombin can cause mucosal damage and tissue dysfunctions. Specific inhibition of thrombin activity, and PAR1 antagonists prevent some intestinal damage in TNBS colitis.ConclusionsIn this study, using both animal models and human IBD tissues, we showed that upregulation of mucosal thrombin alone can lead to inflammatory insults. We propose that targeting downstream events from high thrombin activity, rather than inhibiting thrombin directly, might be a better option for IBD because mucosal thrombin at low dose plays an important role on maintaining tissue homeostasis. Considering these promising preclinical results on PAR1 antagonist, future clinical studies in IBD patients could therefore be rapidly envisioned, particularly in patients with the strongest upregulation of thrombin activity.

Proteolytic homeostasis is important at mucosal surfaces, but its actors and their precise role in physiology are poorly understood. Here we report that healthy human and mouse colon epithelia are a major source of active thrombin. We show that mucosal thrombin is directly regulated by the presence of commensal microbiota. Specific inhibition of luminal thrombin activity causes macroscopic and microscopic damage as well as transcriptomic alterations of genes involved in host-microbiota interactions. Further, luminal thrombin inhibition impairs the spatial segregation of microbiota biofilms, allowing bacteria to invade the mucus layer and to translocate across the epithelium. Thrombin cleaves the biofilm matrix of reconstituted mucosa-associated human microbiota. Our results indicate that thrombin constrains biofilms at the intestinal mucosa. Further work is needed to test whether thrombin plays similar roles in other mucosal surfaces, given that lung, bladder and skin epithelia also express thrombin.

Objective The gut-brain axis is considered as a major regulatory checkpoint in the control of glucose homeostasis. The detection of nutrients and/or hormones in the duodenum informs the hypothalamus of the host's nutritional state. This process may occur via hypothalamic neurons modulating central release of nitric oxide (NO), which in turn controls glucose entry into tissues. The enteric nervous system (ENS) modulates intestinal contractions in response to various stimuli, but the importance of this interaction in the control of glucose homeostasis via the brain is unknown. We studied whether apelin, a bioactive peptide present in the gut, regulates ENS-evoked contractions, thereby identifying a new physiological partner in the control of glucose utilisation via the hypothalamus.Design We measured the effect of apelin on electrical and mechanical duodenal responses via telemetry probes and isotonic sensors in normal and obese/diabetic mice. Changes in hypothalamic NO release, in response to duodenal contraction modulated by apelin, were evaluated in real time with specific amperometric probes. Glucose utilisation in tissues was measured with orally administrated radiolabeled glucose.Results In normal and obese/diabetic mice, glucose utilisation is improved by the decrease of ENS/contraction activities in response to apelin, which generates an increase in hypothalamic NO release. As a consequence, glucose entry is significantly increased in the muscle.Conclusions Here, we identify a novel mode of communication between the intestine and the hypothalamus that controls glucose utilisation. Moreover, our data identified oral apelin administration as a novel potential target to treat metabolic disorders.

OBJECTIVES: Elafin, an endogenous serine protease inhibitor, modulates colonic inflammation. We investigated the role of elafin in celiac disease (CD) using human small intestinal tissues and in vitro assays of gliadin deamidation. We also investwigated the potential beneficial effects of elafin in a mouse model of gluten sensitivity.METHODS: Epithelial elafin expression in the small intestine of patients with active CD, treated CD, and controls without CD was determined by immunofluorescence. Interaction of elafin with human tissue transglutaminase-2 (TG-2) was investigated in vitro. The 33-mer peptide, a highly immunogenic gliadin peptide, was incubated with TG-2 and elafin at different concentrations. The degree of deamidation of the 33-mer peptide was analyzed by liquid chromatography-mass spectrometry. Elafin was delivered to the intestine of gluten-sensitive mice using a recombinant Lactococcus lactis vector. Small intestinal barrier function, inflammation, proteolytic activity, and zonula occludens-1 (ZO-1) expression were assessed.RESULTS: Elafin expression in the small intestinal epithelium was lower in patients with active CD compared with control patients. In vitro, elafin significantly slowed the kinetics of the deamidation of the 33-mer peptide to its more immunogenic form. Treatment of gluten-sensitive mice with elafin delivered by the L. lactis vector normalized inflammation, improved permeability, and maintained ZO-1 expression.CONCLUSIONS: The decreased elafin expression in the small intestine of patients with active CD, the reduction of 33-mer peptide deamidation by elafin, coupled to the barrier enhancing and anti-inflammatory effects observed in gluten-sensitive mice, suggest that this molecule may have pathophysiological and therapeutic importance in gluten-related disorders.

Lipid autacoids derived from n-3/n-6 polyunsaturated fatty acids (PUFA) are some of the earliest signals triggered by an inflammatory reaction. They are acting also as essential regulators of numerous biological processes in physiological conditions. With regards to their importance, a robust and rapid procedure to quantify a large variety of PUFA metabolites, applicable to diverse biological components needed to be formulated. We have developed a simple methodology using liquid chromatography-tandem mass spectrometry allowing quantification of low-level of PUFA metabolites including bioactive mediators, inactive products and pathway biomarkers. Solid phase extraction was used for samples preparation with an extraction yield of 80% ranging from 65% to 98%. The method was optimized to obtain a rapid (8.5 min) and accurate separation of 26 molecules, with a very high sensitivity of detection and analysis (0.6-155 pg). When applied to biological samples, the method enabled characterization of eicosanoids and docosanoids production in epithelial cells or foam macrophages stimulated with LPS, in biological fluids and tissues from mouse models of peritonitis or infectious colitis. Our results demonstrate that this new method can be used in cultured cells, in fluids and in colonic tissues to quantify pro-inflammatory and pro-resolving PUFA metabolites mediators. (C) 2013 Elsevier B.V. All rights reserved.

In contrast, Imatinib did not affect bacterial attachment under standard stationary cell culture conditions, confirming prior observations on the redundant role of ABL and ARG kinases under these circumstances.In further studies, we have identified in library screening experiments several additional promising kinase inhibitors that significantly blocked bacterial attachment to cultured epithelial cells.Several of these inhibitors target kinase pathways not previously known to be involved in EPEC attachment.Together, these data provide new insights into the possible utility of kinase inhibitors in treating and preventing infections with EPEC and perhaps other A/E pathogens.

Elafin, a natural protease inhibitor expressed in healthy intestinal mucosa, has pleiotropic anti-inflammatory properties in vitro and in animal models. We found that mucosal expression of Elafin is diminished in patients with inflammatory bowel disease (IBD). This defect is associated with increased elastolytic activity (elastase-like proteolysis) in colon tissue. We engineered two food-grade strains of lactic acid bacteria (LAB) to express and deliver Elafin to the site of inflammation in the colon to assess the potential therapeutic benefits of the Elafin-expressing LAB. In mouse models of acute and chronic colitis, oral administration of Elafin-expressing LAB decreased elastolytic activity and inflammation and restored intestinal homeostasis. Furthermore, when cultures of human intestinal epithelial cells were treated with LAB secreting Elafin, the inflamed epithelium was protected from increased intestinal permeability and from the release of cytokines and chemokines, both of which are characteristic of intestinal dysfunction associated with IBD. Together, these results suggest that oral delivery of LAB secreting Elafin may be useful for treating IBD in humans.

BACKGROUND & AIMS: Colonic tissues of patients with inflammatory bowel disease have been reported to have increased proteolytic activity, but no studies have clearly addressed the role of the balance between proteases and antiproteases in the pathogenesis of colitis. We investigated the role of Elafin, a serine protease inhibitor expressed by skin and mucosal surfaces in human inflammatory conditions, and the proteases neutrophil elastase (NE) and proteinase-3 (PR-3) in mice with colitis. METHODS: We studied mice with heterozygous disruptions in NE and PR-3, mice that express human elafin (an inhibitor of NE and PR-3), and naive mice that received intracolonic adenoviral vectors that express elafin. Trinitrobenzene sulfonic acid (TNBS) or dextran sodium sulphate (DSS) was used to induce colitis. Protease, cytokine levels, and NF-kappa B activity were measured in colons of mice. Caco-2 and HT29 cells were studied in assays for cytokine expression, permeability, and NF-kappa B activity. RESULTS: Elafin expression or delivery re-equilibrated the proteolytic balance in inflamed colons of mice. In mice given TNBS or DSS, transgenic expression of elafin or disruption of NE and PR-3 protected against the development of colitis. Similarly, adenoviral delivery of Elafin significantly inhibited inflammatory parameters. Elafin modulated a variety of inflammatory mediators in vitro and in vivo and strengthened intestinal epithelial barrier functions. CONCLUSIONS: The protease inhibitor Elafin prevents intestinal inflammation in mouse models of colitis and might be developed as a therapeutic agent for inflammatory bowel disease.

Positive correlations between laboratory-based markers of fibrosis and transient elastography were highly significant.