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Titanium dioxide (TiO2) nanoparticles are widely used in cosmetics, sunscreens, biomedicine, and food products. When used as a food additive, TiO2 nanoparticles are used in significant amounts as white food-coloring agents. However, the effects of TiO2 nanoparticles on the gastrointestinal tract remain unclear. The present study was designed to determine the effects of five TiO2 particles of different crystal structures and sizes in human epithelial colorectal adenocarcinoma (Caco-2) cells and THP-1 monocyte-derived macrophages. Twenty-four-hour exposure to anatase (primary particle size: 50 and 100 nm) and rutile (50 nm) TiO2 particles reduced cellular viability in a dose-dependent manner in THP-1 macrophages, but in not Caco-2 cells. However, 72-h exposure of Caco-2 cells to anatase (50 nm) TiO2 particles reduced cellular viability in a dose-dependent manner. The highest dose (50 µg/mL) of anatase (100 nm), rutile (50 nm), and P25 TiO2 particles also reduced cellular viability in Caco-2 cells. The production of reactive oxygen species tended to increase in both types of cells, irrespective of the type of TiO2 particle. Exposure of THP-1 macrophages to 50 µg/mL of anatase (50 nm) TiO2 particles increased interleukin (IL)-1β expression level, and exposure of Caco-2 cells to 50 µg/mL of anatase (50 nm) TiO2 particles also increased IL-8 expression. The results indicated that anatase TiO2 nanoparticles induced inflammatory responses compared with other TiO2 particles. Further studies are required to determine the in vivo relevance of these findings to avoid the hazards of ingested particles.

SLC16A13, which encodes the monocarboxylate transporter 13 (MCT13), is a susceptibility gene for type 2 diabetes and is expressed in the liver and duodenum. Some peptidase-resistant oligopeptides are absorbed in the gastrointestinal tract and affect glycemic control in the body. Their efficient absorption is mediated by oligopeptide transporter(s) at the apical and basolateral membranes of the intestinal epithelia; however, the molecules responsible for basolateral oligopeptide transport have not been identified. In this study, we examined whether MCT13 functions as a novel basolateral oligopeptide transporter. We evaluated the uptake of oligopeptides and peptidomimetics in MCT13-transfected cells. The uptake of cephradine, a probe for peptide transport system(s), significantly increased in MCT13-transfected cells, and this increase was sensitive to membrane potential. The cellular accumulation of bioactive peptides, such as anserine and carnosine, was decreased by MCT13, indicating MCT13-mediated efflux transport activity. In polarized Caco-2 cells, MCT13 was localized at the basolateral membrane. MCT13 induction enhanced cephradine transport in an apical-to-basal direction across Caco-2 cells. These results indicate that MCT13 functions as a novel efflux transporter of oligopeptides and peptidomimetics, driven by electrochemical gradients across the plasma membrane, and it may be involved in the transport of these compounds across the intestinal epithelia.

ObjectiveHuman intestinal epithelial organoids (IEOs) are increasingly being recognised as a highly promising translational research tool. However, our understanding of their epigenetic molecular characteristics and behaviour in culture remains limited.DesignWe performed genome-wide DNA methylation and transcriptomic profiling of human IEOs derived from paediatric/adult and fetal small and large bowel as well as matching purified human gut epithelium. Furthermore, organoids were subjected to in vitro differentiation and genome editing using CRISPR/Cas9 technology.ResultsWe discovered stable epigenetic signatures which define regional differences in gut epithelial function, including induction of segment-specific genes during cellular differentiation. Established DNA methylation profiles were independent of cellular environment since organoids retained their regional DNA methylation over prolonged culture periods. In contrast to paediatric and adult organoids, fetal gut-derived organoids showed distinct dynamic changes of DNA methylation and gene expression in culture, indicative of an in vitro maturation. By applying CRISPR/Cas9 genome editing to fetal organoids, we demonstrate that this process is partly regulated by TET1, an enzyme involved in the DNA demethylation process. Lastly, generating IEOs from a child diagnosed with gastric heterotopia revealed persistent and distinct disease-associated DNA methylation differences, highlighting the use of organoids as disease-specific research models.ConclusionsOur study demonstrates striking similarities of epigenetic signatures in mucosa-derived IEOs with matching primary epithelium. Moreover, these results suggest that intestinal stem cell-intrinsic DNA methylation patterns establish and maintain regional gut specification and are involved in early epithelial development and disease.

Inflammatory bowel diseases (IBDs), including Crohn’s disease and ulcerative colitis, are associated with dysbiosis of the gut microbiome. Emerging evidence suggests that small-molecule metabolites derived from bacterial breakdown of a variety of dietary nutrients confer a wide array of host benefits, including amelioration of inflammation in IBDs. Yet, in many cases, the molecular pathways targeted by these molecules remain unknown. Here, we describe roles for three metabolites—indole-3-ethanol, indole-3-pyruvate, and indole-3-aldehyde—which are derived from gut bacterial metabolism of the essential amino acid tryptophan, in regulating intestinal barrier function.We determined that these metabolites protect against increased gut permeability associated with a mouse model of colitis by maintaining the integrity of the apical junctional complex and its associated actin regulatory proteins, including myosin IIA and ezrin, and that these effects are dependent on the aryl hydrocarbon receptor. Our studies provide a deeper understanding of how gut microbial metabolites affect host defense mechanisms and identify candidate pathways for prophylactic and therapeutic treatments for IBDs.

The gastrointestinal epithelium forms the boundary between the body and external environment. It effectively provides a selective permeable barrier that limits the permeation of luminal noxious molecules, such as pathogens, toxins, and antigens, while allowing the appropriate absorption of nutrients and water. This selective permeable barrier is achieved by intercellular tight junction (TJ) structures, which regulate paracellular permeability. Disruption of the intestinal TJ barrier, followed by permeation of luminal noxious molecules, induces a perturbation of the mucosal immune system and inflammation, and can act as a trigger for the development of intestinal and systemic diseases. In this context, much effort has been taken to understand the roles of extracellular factors, including cytokines, pathogens, and food factors, for the regulation of the intestinal TJ barrier. Here, I discuss the regulation of the intestinal TJ barrier together with its implications for the pathogenesis of diseases.

Abstract Clostridium difficile is a bacterial pathogen that is the leading cause of nosocomial antibiotic-associated diarrhea and pseudomembranous colitis worldwide. The incidence, severity, mortality and healthcare costs associated with C. difficile infection (CDI) are rising, making C. difficile a major threat to public health. Traditional treatments for CDI involve use of antibiotics such as metronidazole and vancomycin, but disease recurrence occurs in about 30% of patients, highlighting the need for new therapies. The pathogenesis of C. difficile is primarily mediated by the actions of two large clostridial glucosylating toxins, toxin A (TcdA) and toxin B (TcdB). Some strains produce a third toxin, the binary toxin C. difficile transferase, which can also contribute to C. difficile virulence and disease. These toxins act on the colonic epithelium and immune cells and induce a complex cascade of cellular events that result in fluid secretion, inflammation and tissue damage, which are the hallmark features of the disease. In this review, we summarize our current understanding of the structure and mechanism of action of the C. difficile toxins and their role in disease. This review summarizes the structures, molecular mechanisms and physiological responses to the three toxins associated with disease symptoms in Clostridium difficile infection.

Objective Lactobacillus rhamnosus GG (LGG), a probiotic, given by gavage is radioprotective of the mouse intestine. LGG-induced radioprotection is toll-like receptor 2 (TLR2) and cyclooxygenase-2 (COX-2)-dependent and is associated with the migration of COX-2+mesenchymal stem cells (MSCs) from the lamina propria of the villus to the lamina propria near the crypt epithelial stem cells. Our goals were to define the mechanism of LGG radioprotection including identification of the TLR2 agonist, and the mechanism of the MSC migration and to determine the safety and efficacy of this approach in models relevant to clinical radiation therapy.DesignIntestinal radioprotection was modelled in vitro with cell lines and enteroids as well as in vivo by assaying clinical outcomes and crypt survival. Fractionated abdominal and single dose radiation were used along with syngeneic CT26 colon tumour grafts to assess tumour radioprotection.ResultsLGG with a mutation in the processing of lipoteichoic acid (LTA), a TLR2 agonist, was not radioprotective, while LTA agonist and native LGG were. An agonist of CXCR4 blocked LGG-induced MSC migration and LGG-induced radioprotection. LGG given by gavage induced expression of CXCL12, a CXCR4 agonist, in pericryptal macrophages and depletion of macrophages by clodronate liposomes blocked LGG-induced MSC migration and radioprotection. LTA effectively protected the normal intestinal crypt, but not tumours in fractionated radiation regimens.ConclusionsLGG acts as a ‘time-release capsule’ releasing radioprotective LTA. LTA then primes the epithelial stem cell niche to protect epithelial stem cells by triggering a multicellular, adaptive immune signalling cascade involving macrophages and PGE2 secreting MSCs.Trial registration number NCT01790035; Pre-results.

ObjectiveReduced Paneth cell (PC) numbers are observed in inflammatory bowel diseases and impaired PC function contributes to the ileal pathogenesis of Crohn’s disease (CD). PCs reside in proximity to Lgr5+ intestinal stem cells (ISC) and mitochondria are critical for ISC-renewal and differentiation. Here, we characterise ISC and PC appearance under inflammatory conditions and describe the role of mitochondrial function for ISC niche-maintenance.DesignIleal tissue samples from patients with CD, mouse models for mitochondrial dysfunction (Hsp60Δ/ΔISC) and CD-like ileitis (TNFΔARE), and intestinal organoids were used to characterise PCs and ISCs in relation to mitochondrial function.ResultsIn patients with CD and TNFΔARE mice, inflammation correlated with reduced numbers of Lysozyme-positive granules in PCs and decreased Lgr5 expression in crypt regions. Disease-associated changes in PC and ISC appearance persisted in non-inflamed tissue regions of patients with CD and predicted the risk of disease recurrence after surgical resection. ISC-specific deletion of Hsp60 and inhibition of mitochondrial respiration linked mitochondrial function to the aberrant PC phenotype. Consistent with reduced stemness in vivo, crypts from inflamed TNFΔARE mice fail to grow into organoids ex vivo. Dichloroacetate-mediated inhibition of glycolysis, forcing cells to shift to mitochondrial respiration, improved ISC niche function and rescued the ability of TNFΔARE mice-derived crypts to form organoids.ConclusionWe provide evidence that inflammation-associated mitochondrial dysfunction in the intestinal epithelium triggers a metabolic imbalance, causing reduced stemness and acquisition of a dysfunctional PC phenotype. Blocking glycolysis might be a novel drug target to antagonise PC dysfunction in the pathogenesis of CD.

The microbial metabolite butyrate serves as a link between the intestinal microbiome and epithelium. The monocarboxylate transporters MCT1 and SMCT1 are the predominant means of butyrate transport from the intestinal lumen to epithelial cytoplasm, where the molecule undergoes rapid β-oxidation to generate cellular fuel. However, not all epithelial cells metabolize butyrate equally. Undifferentiated colonocytes, including neoplastic cells and intestinal stem cells at the epithelial crypt base preferentially utilize glucose over butyrate for cellular fuel. This divergent metabolic conditioning is central to the phenomenon known as “butyrate paradox”, in which butyrate induces contradictory effects on epithelial proliferation in undifferentiated and differentiated colonocytes. There is evidence that accumulation of butyrate in epithelial cells results in histone modification and altered transcriptional activation that halts cell cycle progression. This manifests in the apparent protective effect of butyrate against colonic neoplasia. A corollary to this process is butyrate-induced inhibition of intestinal stem cells. Yet, emerging research has illustrated that the evolution of the crypt, along with butyrate-producing bacteria in the intestine, serve to protect crypt base stem cells from butyrate’s anti-proliferative effects. Butyrate also regulates epithelial inflammation and tolerance to antigens, through production of anti-inflammatory cytokines and induction of tolerogenic dendritic cells. The role of butyrate in the pathogenesis and treatment of intestinal neoplasia, inflammatory bowel disease and malabsorptive states is evolving, and holds promise for the potential translation of butyrate’s cellular function into clinical therapies.