Human Intestinal Explants Research Articles

Assessing the impact of insect protein sources on intestinal health and disease: insights from human ex vivo and rat in vivo models.

The exploration of edible insects, specifically Alphitobius diaperinus and Tenebrio molitor, as sustainable sources of protein for human consumption is an emerging field. However, research into their effects on intestinal health, especially in relation to inflammation and permeability, remains limited. Using ex vivo and in vivo models of intestinal health and disease, in this study we assess the impact of the above insects on intestinal function by focusing on inflammation, barrier dysfunction and morphological changes. Initially, human intestinal explants were exposed to in vitro-digested extracts of these insects, almond and beef. Immune secretome analysis showed that the inflammatory response to insect-treated samples was comparatively lower than it was for samples exposed to almond and beef. Animal studies using yellow mealworm (Tenebrio molitor) and buffalo (Alphitobius diaperinus) flours were then used to evaluate their safety in healthy rats and LPS-induced intestinal dysfunction rats. Chronic administration of these insect-derived flours showed no adverse effects on behavior, metabolism, intestinal morphology or immune response (such as inflammation or allergy markers) in healthy Wistar rats. Notably, in rats subjected to proinflammatory LPS-induced intestinal dysfunction, T. molitor consumption did not exacerbate symptoms, nor did it increase allergic responses. These findings validate the safety of these edible insects under healthy conditions, demonstrate their innocuity in a model of intestinal dysfunction, and underscore their promise as sustainable and nutritionally valuable dietary protein sources.

Butyrate acts through HDAC inhibition to enhance aryl hydrocarbon receptor activation by gut microbiota-derived ligands

ABSTRACT Aryl hydrocarbon receptor (AhR) is a critical player in the crosstalk between the gut microbiota and its host. However, factors regulating AhR within the gut, which is a complex metabolomic environment, are poorly understood. This study investigates the effect of a combination of metabolites on the activation mechanism of AhR. AhR activity was evaluated using both a luciferase reporter system and mRNA levels of AhR target genes on human cell lines and human colonic explants. AhR activation was studied by radioligand-binding assay, nuclear translocation of AhR by immuofluorescence and protein co-immunoprecipitation of AhR with ARNT. Indirect activation of AhR was evaluated using several tests and inhibitors. The promoter of the target gene CYP1A1 was studied both by chromatin immunoprecipitation and by using an histone deacetylase HDAC inhibitor (iHDAC). Short-chain fatty acids, and butyrate in particular, enhance AhR activity mediated by endogenous tryptophan metabolites without binding to the receptor. This effect was confirmed in human intestinal explants and did not rely on activation of receptors targeted by SCFAs, inhibition of AhR degradation or clearance of its ligands. Butyrate acted directly on AhR target gene promoter to reshape chromatin through iHDAC activity. Our findings revealed that butyrate is not an AhR ligand but acts as iHDAC leading to an increase recruitment of AhR to the target gene promoter in the presence of tryptophan-derived AhR agonists. These data contribute to a novel understanding of the complex regulation of AhR activation by gut microbiota-derived metabolites.

Intestinal explant barrier chip: long-term intestinal absorption screening in a novel microphysiological system using tissue explants

The majority of intestinal in vitro screening models use cell lines that do not reflect the complexity of the human intestinal tract and hence often fail to accurately predict intestinal drug absorption. Tissue explants have intact intestinal architecture and cell type diversity, but show short viability in static conditions. Here, we present a medium throughput microphysiological system, Intestinal Explant Barrier Chip (IEBC), that creates a dynamic microfluidic microenvironment and prolongs tissue viability. Using a snap fit mechanism, we successfully incorporated human and porcine colon tissue explants and studied tissue functionality, integrity and viability for 24 hours. With a proper distinction of transcellular over paracellular transport (ratio >2), tissue functionality was good at early and late timepoints. Low leakage of FITC-dextran and preserved intracellular lactate dehydrogenase levels indicate maintained tissue integrity and viability, respectively. From a selection of low to high permeability drugs, 6 out of 7 properly ranked according to their fraction absorbed. In conclusion, the IEBC is a novel screening platform benefitting from the complexity of tissue explants and the flow in microfluidic chips.

Adding Fuel to the Fire? A Role of Intraepithelial Lymphocytes in Enteric Immune Responses to SARS-CoV-2 Infection

Adding Fuel to the Fire? A Role of Intraepithelial Lymphocytes in Enteric Immune Responses to SARS-CoV-2 Infection

Blockage of bacterial FimH prevents mucosal inflammation associated with Crohn\u2019s disease

BackgroundAn Escherichia coli (E. coli) pathotype with invasive properties, first reported by Darfeuille-Michaud and termed adherent-invasive E. coli (AIEC), was shown to be prevalent in up to half the individuals with Crohn’s Disease (CD), suggesting that these bacteria could be involved in the pathophysiology of CD. Among the genes related to AIEC pathogenicity, fim has the potential to generate an inflammatory reaction from the intestinal epithelial cells and macrophages, as it interacts with TLR4, inducing the production of inflammatory cytokines independently of LPS. Therefore, targeting the bacterial adhesion of FimH-expressing bacteria seems a promising therapeutic approach, consisting of disarming bacteria without killing them, representing a selective strategy to suppress a potentially critical trigger of intestinal inflammation, without disturbing the intestinal microbiota.ResultsWe analyzed the metagenomic composition of the gut microbiome of 358 patients with CD from two different cohorts and characterized the presence of FimH-expressing bacteria. To assess the pathogenic role of FimH, we used human intestinal explants and tested a specific FimH blocker to prevent bacterial adhesion and associated inflammation. We observed a significant and disease activity-dependent enrichment of Enterobacteriaceae in the gut microbiome of patients with CD. Bacterial FimH expression was functionally confirmed in ileal biopsies from 65% of the patients with CD. Using human intestinal explants, we further show that FimH is essential for adhesion and to trigger inflammation. Finally, a specific FimH-blocker, TAK-018, inhibits bacterial adhesion to the intestinal epithelium and prevents inflammation, thus preserving mucosal integrity.ConclusionsWe propose that TAK-018, which is safe and well tolerated in humans, is a promising candidate for the treatment of CD and in particular in preventing its recurrence.Dv5gxXFC6aaMrbE5qenQmhVideo abstract

Human Intestinal Tissue Explant Exposure to Silver Nanoparticles Reveals Sex Dependent Alterations in Inflammatory Responses and Epithelial Cell Permeability.

Consumer products manufactured with antimicrobial silver nanoparticles (AgNPs) may affect the gastrointestinal (GI) system. The human GI-tract is complex and there are physiological and anatomical differences between human and animal models that limit comparisons between species. Thus, assessment of AgNP toxicity on the human GI-tract may require tools that allow for the examination of subtle changes in inflammatory markers and indicators of epithelial perturbation. Fresh tissues were excised from the GI-tract of human male and female subjects to evaluate the effects of AgNPs on the GI-system. The purpose of this study was to perform an assessment on the ability of the ex vivo model to evaluate changes in levels of pro-/anti-inflammatory cytokines/chemokines and mRNA expression of intestinal permeability related genes induced by AgNPs in ileal tissues. The ex vivo model preserved the structural and biological functions of the in-situ organ. Analysis of cytokine expression data indicated that intestinal tissue of male and female subjects responded differently to AgNP treatment, with male samples showing significantly elevated Granulocyte-macrophage colony-stimulating factor (GM-CSF) after treatment with 10 nm and 20 nm AgNPs for 2 h and significantly elevated RANTES after treatment with 20 nm AgNPs for 24 h. In contrast, tissues of female showed no significant effects of AgNP treatment at 2 h and significantly decreased RANTES (20 nm), TNF-α (10 nm), and IFN-γ (10 nm) at 24 h. Smaller size AgNPs (10 nm) perturbed more permeability-related genes in samples of male subjects, than in samples from female subjects. In contrast, exposure to 20 nm AgNPs resulted in upregulation of a greater number of genes in female-derived samples (36 genes) than in male-derived samples (8 genes). The ex vivo tissue model can distinguish sex dependent effects of AgNP and could serve as a translational non-animal model to assess the impacts of xenobiotics on human intestinal mucosa.

Food additives: Assessing the impact of exposure to permitted emulsifiers on bowel and metabolic health - introducing the FADiets study.

Emulsifiers are common components of processed foods consumed as part of a Western diet. Emerging in vitro cell‐line culture, mouse model and human intestinal tissue explant studies have all suggested that very low concentrations of the food emulsifier polysorbate 80 may cause bacterial translocation across the intestinal epithelium, intestinal inflammation and metabolic syndrome. This raises the possibility that dietary emulsifiers might be factors in conditions such as coronary artery disease, type 2 diabetes and Crohn's disease. The potential mechanism behind the observed effects of this emulsifier is uncertain but may be mediated via changes in the gut microbiota or by increased bacterial translocation, or both. It is also unknown whether these effects are generalisable across all emulsifiers and detergents, including perhaps the natural emulsifier lecithin or even conjugated bile acids, particularly if the latter escape reabsorption and pass through to the distal ileum or colon. A major objective of the Medical Research Council (MRC)‐funded Mechanistic Nutrition in Health (MECNUT) Emulsifier project is therefore to investigate the underlying mechanisms and effects of a range of synthetic and natural emulsifiers and detergents in vitro and in vivo, and to determine the effects of a commonly consumed emulsifier (soya lecithin) on gut and metabolic health through a controlled dietary intervention study in healthy human volunteers – the FADiets study. This report provides an overview of the relevant literature, discussing the impact of emulsifiers and other additives on intestinal and metabolic health, and gives an overview of the studies being undertaken as part of the MECNUT Emulsifier project.

The delivery of luminal substances across small intestinal epithelium via goblet cell associated antigen passages is increased in the presence of dietary gliadin.

Abstract Celiac disease is an enteropathy caused by CD4+ T cell responses to dietary gliadin, resulting in damage to the intestinal mucosa and malabsorption. How gliadin peptides cross the epithelium to interact with the immune system is unclear. We used an in vivo imaging approach on live mice and ex vivo assays on mouse and human intestinal explants to evaluate how gliadin traversed the intestinal epithelium. We observed that gliadin crossed the epithelial barrier of mice and humans via the recently described goblet cell associated antigen passages (GAPs), and moreover, gliadin increased the formation of GAPs. The induction of GAPs by gliadin was independent of acetylcholine signaling, the pathway inducing GAP formation in the steady state. Consistent with the increase in GAPs induced by gliadin, LP-DCs isolated from mice given luminal ovalbumin (Ova) concurrently with gliadin induced enhanced antigen specific T cell responses to Ova in ex vivo LP-DC T cell co-culture assays. In vivo Ova specific T cell responses in the MLN were enhanced in mice given Ova concurrent with gliadin. Gliadin did not increase the antigen presentation capacity of the LP-DCs when added to ex vivo cultures, indicating that increased luminal antigen delivery via GAPs mediates the enhanced antigen specific T cell responses to luminal Ova. In the absence of GC’s and GAPs gliadin did not enhance the delivery of luminal antigens to the LP-DCs and did not enhance T cell responses to luminal antigens. These studies identify that gliadin uses GAPs to cross the epithelial barrier, and that gliadin induces further GAP formation in a goblet cell intrinsic manner, thus facilitating the delivery of gliadin and other antigens to the immune system.

17-β Estradiol regulates proglucagon-derived peptide secretion in mouse and human α- and L cells.

Clinical and experimental data indicate a beneficial effect of estrogens on energy and glucose homeostasis associated with improved insulin sensitivity and positive effects on insulin secretion. The aim of the study was to investigate the impact of estrogens on proglucagon-producing cells, pancreatic α cells, and enteroendocrine L cells. The consequences of sexual hormone deprivation were evaluated in ovariectomized mice (ovx). Ovx mice exhibited impaired glucose tolerance during oral glucose tolerance tests (OGTT), which was associated with decreased GLP-1 intestinal and pancreatic secretion and content, an effect that was reversed by estradiol (E2) treatment. Indeed, E2 increased oral glucose-induced GLP-1 secretion in vivo and GLP-1 secretion from primary culture of mouse and human α cells through the activation of all 3 estrogen receptors (ERs), whereas E2-induced GLP-1 secretion from mouse and human intestinal explants occurred only by ERβ activation. Underlying the implication of ERβ, its selective agonist WAY20070 was able to restore glucose tolerance in ovx mice at least partly through plasma GLP-1 increase. We conclude that E2 directly controls both α- and L cells to increase GLP-1 secretion, in addition to its effects on insulin and glucagon secretion, highlighting the potential beneficial role of the estrogenic pathway and, more particularly, of ERβ agonists to prevent type 2 diabetes.

The nitric oxide synthase 2 pathway is targeted by both pro- and anti-inflammatory treatments in the immature human intestine

Background and AimNO synthase 2 (NOS2) was recently identified as one the most overexpressed genes in intestinal samples of premature infants with necrotizing enterocolitis (NEC). NOS2 is widely implicated in the processes of epithelial cell injury/apoptosis and host immune defense but its specific role in inflammation of the immature human intestinal mucosa remains unclear. Interestingly, factors that prevent NEC such as epidermal growth factor (EGF) attenuate the inflammatory response in the mid-gestation human small intestine using serum-free organ culture while drugs that are associated with NEC occurrence such as the non-steroidal anti-inflammatory drug, indomethacin (INDO), exert multiple detrimental effects on the immature human intestine. In this study we investigate the potential role of NOS2 in modulating the gut inflammatory response under protective and stressful conditions by determining the expression profile of NOS2 and its downstream pathways in the immature intestine. MethodsGene expression profiles of cultured mid-gestation human intestinal explants were investigated in the absence or presence of a physiological concentration of EGF (50 ng/ml) or 1 μM INDO for 48 h using Illumina whole genome microarrays, Ingenuity Pathway Analysis software and quantitative PCR to investigate the expression of NOS2 and NOS2-pathway related genes. ResultsIn the immature intestine, NOS2 expression was found to be increased by EGF and repressed by INDO. Bioinformatic analysis identified differentially regulated pathways where NOS2 is known to play an important role including citrulline/arginine metabolism, epithelial cell junctions and oxidative stress. At the individual gene level, we identified many differentially expressed genes of the citrulline/arginine metabolism pathway such as ARG1, ARG2, GLS, OAT and OTC in response to EGF and INDO. Gene expression of tight junction components such as CLDN1, CLDN2, CLDN7 and OCN and of antioxidant markers such as DUOX2, GPX2, SOD2 were also found to be differentially modulated by EGF and INDO. ConclusionThese results suggest that the protective effect of EGF and the deleterious influence of INDO on the immature intestine could be mediated via regulation of NOS2. Pathways downstream of NOS2 involved with these effects include metabolism linked to NO production, epithelial barrier permeability and antioxidant expression. These results suggest that NOS2 is a likely regulator of the inflammatory response in the immature human gut and may provide a mechanistic basis for the protective effect of EGF and the deleterious effects of INDO.

Mo1271 Wheat Alpha-Amylase/Trypsin Inhibitors Are Strong Activators of Innate Immunity in Murine and Human Intestinal Tissue Explants

Mo1271 Wheat Alpha-Amylase/Trypsin Inhibitors Are Strong Activators of Innate Immunity in Murine and Human Intestinal Tissue Explants

Subversion of Human Intestinal Mucosa Innate Immunity by a Crohn's Disease‐associated E. coli

Adherent‐invasive Escherichia coli (AIEC), associated with Crohn's disease, are likely candidate contributory factors in the disease. However, signaling pathways involved in human intestinal mucosa innate host response to AIEC remain unknown. Here we use a 3‐D model of human intestinal mucosa explant culture to explore the effects of the AIEC strain LF82, on two innate immunity platforms, i.e. the inflammasome through evaluation of caspase‐1 status, and NFkB signaling. We showed that LF82 bacteria enter and survive within a few intestinal epithelial cells and lamina propria macrophages, without altering the mucosa overall architecture. While 4h infection with a Salmonella strain caused crypt disorganization, caspase‐1 activation and mature IL‐18 production, LF82 bacteria were unable to activate caspase‐1 and induce IL‐18 production. In parallel, LF82 bacteria activated NFkB signaling in epithelial cells through IkBα phosphorylation, NFkBp65 nuclear translocation and TNFα secretion. In addition, pharmacological blockade of NFkB activation using the SN50 peptide showed that NFkB activation was crucial for the maintenance of epithelial homeostasis upon LF82 infection. In conclusion, here we decipher at the whole mucosa level the mechanisms of the LF82‐induced subversion of innate immunity that, by maintaining host cell integrity, ensure intracellular bacteria survival.

Subversion of human intestinal mucosa innate immunity by a Crohn's disease-associated E. coli.

Adherent-invasive Escherichia coli (AIEC), associated with Crohn's disease, are likely candidate contributory factors in the disease. However, signaling pathways involved in human intestinal mucosa innate host response to AIEC remain unknown. Here we use a 3D model of human intestinal mucosa explant culture to explore the effects of the AIEC strain LF82 on two innate immunity platforms, i.e., the inflammasome through evaluation of caspase-1 status, and NFκB signaling. We showed that LF82 bacteria enter and survive within a few intestinal epithelial cells and macrophages, without altering the mucosa overall architecture. Although 4-h infection with a Salmonella strain caused crypt disorganization, caspase-1 activation, and mature IL-18 production, LF82 bacteria were unable to activate caspase-1 and induce IL-18 production. In parallel, LF82 bacteria activated NFκB signaling in epithelial cells through IκBα phosphorylation, NFκBp65 nuclear translocation, and TNFα secretion. In addition, NFκB activation was crucial for the maintenance of epithelial homeostasis upon LF82 infection. In conclusion, here we decipher at the whole-mucosa level the mechanisms of the LF82-induced subversion of innate immunity that, by maintaining host cell integrity, ensure intracellular bacteria survival.

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food. Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out. To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area. We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

Sa2012 Risk Factors and Temporal Trends for Complicated Diverticulitis: A Population-Based Study

Background: Indomethacin (INDO) is a non-steroidal anti-inflammatory drug (NSAID) used for the treatment of patent ductus arteriosus in preterm infants and as a tocolytic agent in pregnant women. The exposure of preterm infants to INDO is associated with an increased risk of developing enteropathies such as necrotizing enterocolitis. Despite increasing progress in establishing the pathways involved in NSAID-induced damage, understanding the pathogenesis of gastrointestinal injury by NSAIDs in neonates relies on a limited number of studies carried out in animal models. Aims: In the present study, we combined our method for maintaining the mid-gestation human intestine in serum-free organ culture with highthroughput genomic studies and bioinformatic pathway analysis to investigate the specific intestinal effects of INDO on the overall physiology of both the small and large intestine at mid-gestation. Methods: After determining the optimal concentration of INDO to be 1μM (.90% inhibition of intestinal prostaglandin E2 production), a dose that corresponds to the range of circulating levels in treated preterm babies, we used Illumina microarrays to establish global gene expression profiles in human intestinal explants (ileums and colons) in response to an INDO treatment of 48 h. Differentially expressed genes between control and INDO-treated explants were determined using the Mann-Whitney test (p , 0.05), and further analyzed with Ingenuity Pathway Analysis software (IPA 8.8) to identify biological functions and canonical pathways modulated by INDO. Results: The gene expression profiles identified revealed that a large proportion of statistically significant biological, physiological and metabolic functions were modulated by INDO in the immature intestine. As expected, a number of these significantly modulated functions were inflammation-related such as Inflammatory diseases, Inflammatory response, and Acute phase response. Noteworthily, we have identified a series of metabolic-related gene clusters that were found to be significantly modulated, namely Glycolysis/gluconeogenesis, Oxidative phosphorylation and Free radical scavenging/oxidoreductase activity. Further analysis by qPCR confirmed that the expression of representative genes involved in these metabolic functions, such as ALDOA, LDHA, NDUFA9, DUOX2, SOD2, and TFF1, was inhibited by INDO in both intestinal segments, indicating that this adverse metabolic effect could be a component of the pathogenesis of intestinal damage induced by INDO. Conclusion: This study identified several different metabolic pathways that are modulated by INDO in the immature human intestine and suggests a mechanistic basis for better understanding the contributing effects of INDO and other NSAIDs on neonatal intestinal pathologies.

Sa2011 Adverse Metabolic Effects of Indomethacin on the Immature Human Intestine

Background: Indomethacin (INDO) is a non-steroidal anti-inflammatory drug (NSAID) used for the treatment of patent ductus arteriosus in preterm infants and as a tocolytic agent in pregnant women. The exposure of preterm infants to INDO is associated with an increased risk of developing enteropathies such as necrotizing enterocolitis. Despite increasing progress in establishing the pathways involved in NSAID-induced damage, understanding the pathogenesis of gastrointestinal injury by NSAIDs in neonates relies on a limited number of studies carried out in animal models. Aims: In the present study, we combined our method for maintaining the mid-gestation human intestine in serum-free organ culture with highthroughput genomic studies and bioinformatic pathway analysis to investigate the specific intestinal effects of INDO on the overall physiology of both the small and large intestine at mid-gestation. Methods: After determining the optimal concentration of INDO to be 1μM (.90% inhibition of intestinal prostaglandin E2 production), a dose that corresponds to the range of circulating levels in treated preterm babies, we used Illumina microarrays to establish global gene expression profiles in human intestinal explants (ileums and colons) in response to an INDO treatment of 48 h. Differentially expressed genes between control and INDO-treated explants were determined using the Mann-Whitney test (p , 0.05), and further analyzed with Ingenuity Pathway Analysis software (IPA 8.8) to identify biological functions and canonical pathways modulated by INDO. Results: The gene expression profiles identified revealed that a large proportion of statistically significant biological, physiological and metabolic functions were modulated by INDO in the immature intestine. As expected, a number of these significantly modulated functions were inflammation-related such as Inflammatory diseases, Inflammatory response, and Acute phase response. Noteworthily, we have identified a series of metabolic-related gene clusters that were found to be significantly modulated, namely Glycolysis/gluconeogenesis, Oxidative phosphorylation and Free radical scavenging/oxidoreductase activity. Further analysis by qPCR confirmed that the expression of representative genes involved in these metabolic functions, such as ALDOA, LDHA, NDUFA9, DUOX2, SOD2, and TFF1, was inhibited by INDO in both intestinal segments, indicating that this adverse metabolic effect could be a component of the pathogenesis of intestinal damage induced by INDO. Conclusion: This study identified several different metabolic pathways that are modulated by INDO in the immature human intestine and suggests a mechanistic basis for better understanding the contributing effects of INDO and other NSAIDs on neonatal intestinal pathologies.

Transintestinal Cholesterol Excretion Is an Active Metabolic Process Modulated by PCSK9 and Statin Involving ABCB1

Transintestinal cholesterol excretion (TICE) is an alternate pathway to hepatobiliary secretion. Our study aimed at identifying molecular mechanisms of TICE. We studied TICE ex vivo in mouse and human intestinal explants, and in vivo after bile diversion and intestinal cannulation in mice. We provide the first evidence that both low-density lipoprotein (LDL) and high-density lipoprotein deliver cholesterol for TICE in human and mouse jejunal explants at the basolateral side. Proprotein convertase subtilisin kexin type 9 (PCSK9)(-/-) mice and intestinal explants show increased LDL-TICE, and acute injection of PCSK9 decreases TICE in vivo, suggesting that PCSK9 is a repressor of TICE. The acute repression was dependent on the LDL receptor (LDLR). Further, TICE was increased when mice were treated with lovastatin. These data point to an important role for LDLR in TICE. However, LDLR(-/-) mice showed increased intestinal LDL uptake, contrary to what is observed in the liver, and tended to have higher TICE. We interpret these data to suggest that there might be at least 2 mechanisms contributing to TICE; 1 involving LDL receptors and other unidentified mechanisms. Acute modulation of LDLR affects TICE, but chronic deficiency is compensated for most likely by the upregulation of the unknown mechanisms. Using mice deficient for apical multidrug active transporter ATP-binding cassette transporter B1 a and b, and its inhibitor, we show that these apical transporters contribute significantly to TICE. TICE is operative in human jejunal explants. It is a metabolically active process that can be acutely regulated, inversely related to cholesterolemia, and pharmacologically activated by statins.

Should probiotics be tested on ex vivo organ culture models?

The use of probiotic strains as nutritional supplements has been gaining ground in the last decade. As the mechanisms with which they modulate innate and adaptive immunity start to unravel, probiotics have repeatedly been suggested as potential treatment for a wide variety of diseases, including inflammatory bowel disease (IBD). However, even though the benefits of probiotic treatment for conditions like atopic dermatitis are well established, very limited clinical benefit has been obtained on IBD treatment. This could be due to the lack of suitable models on which to obtain valid pre-clinical data to select the most appropriate strain for a given condition. We recently described a newly developed model for the culture and apical stimulation of whole human intestinal mucosal explants. We showed that the tissue was only viable if incubated in an O2 chamber, but it was possible to stimulate the tissue with bacteria in a conventional incubator. We used the new set-up to test three different Lactobacilli strains, none of which appeared to be benign on inflamed IBD mucosa.

Extracellular ATP induces cell death in human intestinal epithelial cells

BackgroundExtracellular ATP is an endogenous signaling molecule released by various cell types and under different stimuli. High concentrations of ATP released into the extracellular medium activate the P2X7 receptor in most inflammatory conditions. Here, we seek to characterize the effects of ATP in human intestinal epithelial cells and to evaluate morphological changes in these cells in the presence of ATP. MethodsWe treated human intestinal epithelial cells with ATP and evaluated the effects of this nucleotide by scanning and transmission electron microscopy analysis and calcium measurements. We used flow cytometry to evaluate apoptosis. We collected human intestinal explants for immunohistochemistry, apoptosis by the TUNEL approach and caspase-3 activity using flow cytometry analyses. We also evaluated the ROS production by flow cytometry and NO secretion by the Griess technique. ResultsATP treatment induced changes characteristic of cell death by apoptosis and autophagy but not necrosis in the HCT8 cell line. ATP induced apoptosis in human intestinal explants that showed TUNEL-positive cells in the epithelium and in the lamina propria. The explants exhibited a significant increase of caspase-3 activity when the colonic epithelial cells were incubated with IFN-gamma followed by ATP as compared to control cells. In addition, it was found that antioxidants were able to inhibit both the ROS production and the apoptosis induced by ATP in epithelial cells. General significanceThe activation of P2X7 receptors by ATP induces apoptosis and autophagy in human epithelial cells, possibly via ROS production, and this effect might have implications for gut inflammatory conditions.