Ontogeny and function of the circadian clock in intestinal organoids.

Circadian rhythms regulate diverse aspects of gastrointestinal physiology ranging from the composition of microbiota to motility. However, development of the intestinal circadian clock and detailed mechanisms regulating circadian physiology of the intestine remain largely unknown. In this report, we show that both pluripotent stem cell-derived human intestinal organoids engrafted into mice and patient-derived human intestinal enteroids possess circadian rhythms and demonstrate circadian phase-dependent necrotic cell death responses to Clostridium difficile toxin B (TcdB). Intriguingly, mouse and human enteroids demonstrate anti-phasic necrotic cell death responses to TcdB. RNA-Seq analysis shows that ~3-10% of the detectable transcripts are rhythmically expressed in mouse and human enteroids. Remarkably, we observe anti-phasic gene expression of Rac1, a small GTPase directly inactivated by TcdB, between mouse and human enteroids, and disruption of Rac1 abolishes clock-dependent necrotic cell death responses. Our findings uncover robust functions of circadian rhythms regulating clock-controlled genes in both mouse and human enteroids governing organism-specific, circadian phase-dependent necrotic cell death responses, and lay a foundation for human organ- and disease-specific investigation of clock functions using human organoids for translational applications.

MLL1 is required for maintenance of intestinal stem cells.

Epigenetic mechanisms are gatekeepers for the gene expression patterns that establish and maintain cellular identity in mammalian development, stem cells and adult homeostasis. Amongst many epigenetic marks, methylation of histone 3 lysine 4 (H3K4) is one of the most widely conserved and occupies a central position in gene expression. Mixed lineage leukemia 1 (MLL1/KMT2A) is the founding mammalian H3K4 methyltransferase. It was discovered as the causative mutation in early onset leukemia and subsequently found to be required for the establishment of definitive hematopoiesis and the maintenance of adult hematopoietic stem cells. Despite wide expression, the roles of MLL1 in non-hematopoietic tissues remain largely unexplored. To bypass hematopoietic lethality, we used bone marrow transplantation and conditional mutagenesis to discover that the most overt phenotype in adult Mll1-mutant mice is intestinal failure. MLL1 is expressed in intestinal stem cells (ISCs) and transit amplifying (TA) cells but not in the villus. Loss of MLL1 is accompanied by loss of ISCs and a differentiation bias towards the secretory lineage with increased numbers and enlargement of goblet cells. Expression profiling of sorted ISCs revealed that MLL1 is required to promote expression of several definitive intestinal transcription factors including Pitx1, Pitx2, Foxa1, Gata4, Zfp503 and Onecut2, as well as the H3K27me3 binder, Bahcc1. These results were recapitulated using conditional mutagenesis in intestinal organoids. The stem cell niche in the crypt includes ISCs in close association with Paneth cells. Loss of MLL1 from ISCs promoted transcriptional changes in Paneth cells involving metabolic and stress responses. Here we add ISCs to the MLL1 repertoire and observe that all known functions of MLL1 relate to the properties of somatic stem cells, thereby highlighting the suggestion that MLL1 is a master somatic stem cell regulator.

Overexpression of miRNA29a gene inhibits proliferation and promotes apoptosis of jejunal epithelial cells in yak.

MicroRNAs (miRNAs) were identified to be involved in various biological functions by regulating the degradation or suppressing the translation of their downstream target genes. Recent studies have identified miR-29a play a key role in functions of mammal cell differentiation, proliferation, apoptosis, and signal transduction. However, the underlying functions for miR-29a in jejunal epithelial cells biological function still to be investigated. In order to explore the yak jejunal epithelial cells proliferation and barrier dysfunction with over expression of miR-29a gene, three 0-day-old Pamir male yaks were randomly selected and slaughtered in present study, and the jejunal epithelial cells were isolated and cultured to determine yak jejunal epithelial cells proliferation and protein composition on differential expression of miR-29a gene in Pamir plateau. Here, we demonstrated that the overexpression of miR-29a gene could inhibit the proliferation of Pamir yaks jejunum epithelial cells, and contribute to the apoptosis of Pamir yaks jejunal epithelial cells with some extent. A total of 133 differentially expressed proteins were identified in different expression of miR-29a groups by label-free Mass Spectrometry (MS), which could be concluded to two predominant themes: cell proliferation and inflammatory response. Interestingly, GPR41, as a bridge protein, was contacted two predominant themes to involved in Pamir Yaks jejunal mechanical barrier PPI network, and the target proteins displayed strong mutual interactions in the complex PPI network. Overall, our study suggested that the over-expression miR-29a inhibited the jejunal epithelial cells proliferation and the expressions of specific proteins, which damaged jejunal barrier function to slow down the intestine structure and function advanced mature development during young livestock period for influence the enhanced performance of production efficiency.

Mechanism of endocytic regulation of intestinal tight junction remodeling during nutrient starvation in jejunal IPEC-J2 cells.

Intestinal epithelial cells are tightly bound by tight junction proteins (TJP) which are dynamic and sensitive to environmental stress. However, the role of the endocytic pathway in the regulation of TJP abundance and tight junction integrity during nutrient stress is poorly understood. Therefore, this study was conducted to investigate the regulation of TJP abundance during nutrient starvation and the role of the endocytic mechanism in this process. IPEC-J2 cells were subjected to nutrient starvation in Krebs-Ringer bicarbonate buffer (KRB) and abundance of TJP, an indication of tight junction remodeling, was characterized with RT-PCR, western blotting and immunofluorescence. Abundance of TJP was dynamically regulated by nutrient starvation. The protein levels of claudin-1, 3, and 4 were initially downregulated within the first 6 hours of starvation, and then, increased thereafter (P < .01). However, there was no change in occludin and ZO-1. Lysosome and proteasome inhibitors were used to determine the contribution of these protein degradation pathways to the TJP remodeling. Short-term starvation-induced degradation of claudin-1, 3, and 4 was found to be lysosome dependent. Specifically, the downregulation of claudin-3 and 4 was via a dynamin-dependent, but clathrin and caveolae independent, endocytic pathway and this downregulation was partly reversed by amino acids supplementation. Interestingly, the re-synthesis of TJP with prolonged starvation partly depended on proteasome function. Collectively, this study, for the first time, elucidated a major role for dynamin-dependent endocytosis of claudin-3 and 4 during nutrient stress in intestinal epithelial cells. Therefore, transient endocytosis inhibition may be a potential mechanism for preserving tight junction integrity and function in metabolic or pathological states such as inflammatory bowel disease that involves destruction of intestinal epithelial TJP.

Absence of the C5a Receptor C5aR2 Worsens Ischemic Tissue Injury by Increasing C5aR1-Mediated Neutrophil Infiltration.

Neutrophil infiltration to ischemic tissues following reperfusion worsens injury. A key driver of neutrophil recruitment and activation is the complement factor C5a, which signals through two receptors, C5aR1 and C5aR2. In this study, we used a neutrophil-dependent mouse model of intestinal ischemia-reperfusion (IR) injury to investigate the underexplored role of C5aR2 in neutrophil mobilization, recruitment, and disease outcomes. We show that intestinal IR induces rapid neutrophil mobilization along with a concomitant reduction in plasma C5a levels that is driven by both C5aR1 and C5aR2. Intestinal IR in C5aR2-/- mice led to worsened intestinal damage and increased neutrophil infiltration. Inhibition of C5aR1 signaling in C5aR2-/- mice with PMX53 prevented neutrophil accumulation and reduced IR pathology, suggesting a key requirement for enhanced neutrophil C5aR1 activation in the absence of C5aR2 signaling. Interestingly, C5aR2 deficiency also reduced circulating neutrophil numbers after IR, as well as following G-CSF-mediated bone marrow mobilization, which was independent of C5aR1, demonstrating that C5aR2 has unique and distinct functions from C5aR1 in neutrophil egress. Despite enhanced tissue injury in C5aR2-/- IR mice, there were significant reductions in intestinal proinflammatory cytokines, highlighting complicated dual protective/pathogenic roles for C5aR2 in pathophysiology. Collectively, we show that C5aR2 is protective in intestinal IR by inhibiting C5aR1-mediated neutrophil recruitment to the ischemic tissue. This is despite the potentially local pathogenic effects of C5aR2 in increasing intestinal proinflammatory cytokines and enhancing circulating neutrophil numbers in response to mobilizing signals. Our data therefore suggest that this balance between the dual pro- and anti-inflammatory roles of C5aR2 ultimately dictates disease outcomes.

Porcine epidemic diarrhea virus infection induces endoplasmic reticulum stress and unfolded protein response in jejunal epithelial cells of weaned pigs.

Porcine epidemic diarrhea virus (PEDV) infection leads to diarrhea and subsequently to decreased feed efficiency and growth in weaned pigs. Given that few studies have addressed the host-virus interaction in vivo, this study focused on endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in jejunal epithelial cells during PEDV infection. Eight-week-old pigs (n = 64) were orally inoculated with PEDV IN19338 strain (n = 40) or sham-inoculated (n = 24) and analyzed for PEDV viral RNA shedding using reverse transcription-quantitative polymerase chain reaction and for viral antigen within enterocytes using immunohistochemistry (IHC). ER stress was analyzed in a subset of 9 PEDV-inoculated pigs with diarrhea, detectable viral RNA, and viral antigen (PEDV-immunopositive pigs). Compared with control pigs, PEDV-immunopositive pigs had a reduced ratio of villus height to crypt depth in the jejunum (P = .002, n = 9 per group), consistent with intestinal injury. The protein levels of ATF6, IRE1, PERK, XBP1u, ATF4, GRP78, and caspase-3 were assessed in jejunal epithelial cells at the villus tips via IHC. Both ER stress and UPR were demonstrated in PEDV-immunopositive pigs by the increased expression of ATF6 (P = .047), IRE1 (P = .007), and ATF4 (P = .001). The expression of GRP78 (P = .024) and caspase-3 (P = .004) were also increased, indicating an accompanying increase in ER protein folding capacity and apoptosis. Overall, these results reveal that PEDV infection induces ER stress and UPR in intestinal epithelial cells of weaned pigs.

Infection by the parasitic helminth Trichinella spiralis activates a Tas2r-mediated signaling pathway in intestinal tuft cells.

The parasitic helminth Trichinella spiralis, which poses a serious health risk to animals and humans, can be found worldwide. Recent findings indicate that a rare type of gut epithelial cell, tuft cells, can detect the helminth, triggering type 2 immune responses. However, the underlying molecular mechanisms remain to be fully understood. Here we show that both excretory-secretory products (E-S) and extract of T. spiralis can stimulate the release of the cytokine interleukin 25 (IL-25) from the mouse small intestinal villi and evoke calcium responses from tuft cells in the intestinal organoids, which can be blocked by a bitter-taste receptor inhibitor, allyl isothiocyanate. Heterologously expressed mouse Tas2r bitter-taste receptors, the expression of which is augmented during tuft-cell hyperplasia, can respond to the E-S and extract as well as to the bitter compound salicin whereas salicin in turn can induce IL-25 release from tuft cells. Furthermore, abolishment of the G-protein γ13 subunit, application of the inhibitors for G-protein αo/i, Gßγ subunits, and phospholipase Cß2 dramatically reduces the IL-25 release. Finally, tuft cells are found to utilize the inositol triphosphate receptor type 2 (Ip3r2) to regulate cytosolic calcium and thus Trpm5 activity, while potentiation of Trpm5 by a sweet-tasting compound, stevioside, enhances tuft cell IL-25 release and hyperplasia in vivo. Taken together, T. spiralis infection activates a signaling pathway in intestinal tuft cells similar to that of taste-bud cells, but with some key differences, to initiate type 2 immunity.

Water intake releases serotonin from enterochromaffin cells in rat jejunal villi.

The present study aims to investigate the roles of water intake in serotonin production and release in rat jejunum. We evaluated the changes in concentrations of serotonin in the portal vein and mesenteric lymph vessel induced by the intragastric administration of distilled water. The density of granules in enterochromaffin cells and the immunoreactivity of serotonin in the jejunal villi were investigated before and after water intake. The effects of intravenous administration of serotonin and/or ketanserin on mesenteric lymph flow and concentrations of albumin and IL-22 in the lymph were also addressed. Water intake increased serotonin concentration in the portal vein, but not in the mesenteric lymph vessel. The flux of serotonin through the portal vein was significantly larger than that through the mesenteric lymph vessel. Water intake decreased the density of granules in the enterochromaffin cells and increased the immunoreactivity of serotonin in the jejunal villi. The intravenous administration of serotonin increased significantly mesenteric lymph flow and the concentrations of albumin and IL-22; both were significantly reduced by the intravenous pretreatment with ketanserin. We showed that serotonin released from enterochromaffin cells by water intake was mainly transported through the portal vein. Additionally, serotonin in blood was found to increase mesenteric lymph formation with permeant albumin in the jejunal villi via the activation of 5-HT2 receptor.

Fluid shear stress enhances differentiation of jejunal human enteroids in Intestine-Chip.

There is increasing evidence that the study of normal human enteroids duplicates many known aspects of human intestinal physiology. However, this epithelial cell-only model lacks the many nonepithelial intestinal cells present in the gastrointestinal tract and exposure to the mechanical forces to which the intestine is exposed. We tested the hypothesis that physical shear forces produced by luminal and blood flow would provide an intestinal model more closely resembling normal human jejunum. Jejunal enteroid monolayers were studied in the Emulate, Inc. Intestine-Chip under conditions of constant luminal and basolateral flow that was designed to mimic normal intestinal fluid flow, with human umbilical vein endothelial cells (HUVECs) on the basolateral surface and with Wnt3A, R-spondin, and Noggin only on the luminal surface. The jejunal enteroids formed monolayers that remained confluent for 6-8 days, began differentiating at least as early as day 2 post plating, and demonstrated continuing differentiation over the entire time of the study, as shown by quantitative real-time polymerase chain reaction and Western blot analysis. Differentiation impacted villus genes and proteins differently with early expression of regenerating family member 1α (REG1A), early reduction to a low but constant level of expression of Na+-K+-2Cl- cotransporter 1 (NKCC1), and increasing expression of sucrase-isomaltase (SI) and downregulated in adenoma (DRA). These results were consistent with continual differentiation, as was shown to occur in mouse villus enterocytes. Compared with differentiated enteroid monolayers grown on Transwell inserts, enteroids exposed to flow were more differentiated but exhibited increased apoptosis and reduced carbohydrate metabolism, as shown by proteomic analysis. This study of human jejunal enteroids-on-chip suggests that luminal and basolateral flow produce a model of continual differentiation over time and NaCl absorption that mimics normal intestine and should provide new insights in intestinal physiology.NEW & NOTEWORTHY This study showed that polarized enteroid models in which there is no basolateral Wnt3a, are differentiated, regardless of the Wnt3a status of the apical media. The study supports the concept that in the human intestine villus differentiation is not an all or none phenomenon, demonstrating that at different days after lack of basolateral Wnt exposure, clusters of genes and proteins exist geographically along the villus with different domains having different functions.

Type 2 diabetes is associated with impaired jejunal enteroendocrine GLP-1 cell lineage in human obesity.

OBJECTIVES: Altered enteroendocrine cell (EEC) function in obesity and type 2 diabetes is not fully understood. Understanding the transcriptional program that controls EEC differentiation is important because some EEC types harbor significant therapeutic potential for type 2 diabetes. METHODS: EEC isolation from jejunum of obese individuals with (ObD) or without (Ob) type 2 diabetes was obtained with a new method of cell sorting. EEC transcriptional profiles were established by RNA-sequencing in a first group of 14 Ob and 13 ObD individuals. EEC lineage and densities were studied in the jejunum of a second independent group of 37 Ob, 21 ObD and 22 non obese (NOb) individuals. RESULTS: The RNA seq analysis revealed a distinctive transcriptomic signature and a decreased differentiation program in isolated EEC from ObD compared to Ob individuals. In the second independent group of ObD, Ob and NOb individuals a decreased GLP-1 cell lineage and GLP-1 maturation from proglucagon, were observed in ObD compared to Ob individuals. Furthermore, jejunal density of GLP-1-positive cells was significantly reduced in ObD compared to Ob individuals. CONCLUSIONS: These results highlight that the transcriptomic signature of EEC discriminate obese subjects according to their diabetic status. Furthermore, type 2 diabetes is associated with reduced GLP-1 cell differentiation and proglucagon maturation leading to low GLP-1-cell density in human obesity. These mechanisms could account for the decrease plasma GLP-1 observed in metabolic diseases.

Mechanisms of deoxynivalenol-induced endocytosis and degradation of tight junction proteins in jejunal IPEC-J2 cells involve selective activation of the MAPK pathways.

Mycotoxin contamination in foods is a major risk factor for human and animal health due to its prevalence in cereals and their by-products. Deoxynivalenol (DON), mainly produced by Fusarium genera, is the most common mycotoxin detected in cereal products. Deoxynivalenol disrupts intestinal barrier function and decreases protein levels of tight junction proteins (TJP). However, the overall mechanism by which DON regulates specific TJP turnover and epithelial cell integrity remains unclear. Herein, we show that DON (2 µM) decreases the protein stability and accelerates the degradation of TJP in the lysosome. Interestingly, pretreatment of cells with dynasore (a dynamin-dependent endocytosis inhibitor) protected against DON-induced degradation of claudin-3 and 4. Immunofluorescence analysis also shows that the decreased membrane presence of claudin-4 and ZO-1 induced by DON is reversible with dynamin inhibition, whereas the pretreatment with cytochalasin D (an actin-dependent endocytosis inhibitor) reverses the degradation of claudin-1 and 4 induced by DON. We also show that the endocytosis and degradation of claudin-1 is regulated by p38 mitogen-activated protein kinase (MAPK), whereas the endocytosis of claudin-4 and ZO-1 is mediated by c-Jun-N-terminal kinase (JNK). Resveratrol, with JNK inhibitory activity, also prevents the endocytosis and degradation of claudin-4 and ZO-1 and protects against DON-induced decrease in transepithelial electrical resistance (TEER) and increase in FITC-dextran permeability. Collectively, this study, for the first time, shows that DON accelerates the endocytosis and degradation of TJP and this is regulated by the activation of p38 MAPK and JNK signaling pathways. Therefore, natural bioactive compounds with p38 MAPK and JNK inhibitory activities may be effective in preventing the DON-induced TJP disruption and preserve gut barrier function in vivo.

A population of gut epithelial enterochromaffin cells is mechanosensitive and requires Piezo2 to convert force into serotonin release.

Enterochromaffin (EC) cells constitute the largest population of intestinal epithelial enteroendocrine (EE) cells. EC cells are proposed to be specialized mechanosensory cells that release serotonin in response to epithelial forces, and thereby regulate intestinal fluid secretion. However, it is unknown whether EE and EC cells are directly mechanosensitive, and if so, what the molecular mechanism of their mechanosensitivity is. Consequently, the role of EE and EC cells in gastrointestinal mechanobiology is unclear. Piezo2 mechanosensitive ion channels are important for some specialized epithelial mechanosensors, and they are expressed in mouse and human EC cells. Here, we use EC and EE cell lineage tracing in multiple mouse models to show that Piezo2 is expressed in a subset of murine EE and EC cells, and it is distributed near serotonin vesicles by superresolution microscopy. Mechanical stimulation of a subset of isolated EE cells leads to a rapid inward ionic current, which is diminished by Piezo2 knockdown and channel inhibitors. In these mechanosensitive EE cells force leads to Piezo2-dependent intracellular Ca2+ increase in isolated cells as well as in EE cells within intestinal organoids, and Piezo2-dependent mechanosensitive serotonin release in EC cells. Conditional knockout of intestinal epithelial Piezo2 results in a significant decrease in mechanically stimulated epithelial secretion. This study shows that a subset of primary EE and EC cells is mechanosensitive, uncovers Piezo2 as their primary mechanotransducer, defines the molecular mechanism of their mechanotransduction and mechanosensitive serotonin release, and establishes the role of epithelial Piezo2 mechanosensitive ion channels in regulation of intestinal physiology.

Mechanical Stimuli Affect Escherichia coli Heat-Stable Enterotoxin-Cyclic GMP Signaling in a Human Enteroid Intestine-Chip Model.

Modeling host-pathogen interactions with human intestinal epithelia using enteroid monolayers on permeable supports (such as Transwells) represents an alternative to animal studies or use of colon cancer-derived cell lines. However, the static monolayer model does not expose epithelial cells to mechanical forces normally present in the intestine, including luminal flow and serosal blood flow (shear force) or peristaltic forces. To determine the contribution of mechanical forces in the functional response of human small intestine to a virulence factor of a pathogenic intestinal bacterium, human jejunal enteroids were cultured as monolayers in microengineered fluidic-based Organ-Chips (Intestine-Chips) exposed to enterotoxigenic Escherichia coli heat-stable enterotoxin A (ST) and evaluated under conditions of static fluid, apical and basolateral flow, and flow plus repetitive stretch. Application of flow increased epithelial cell height and apical and basolateral secretion of cyclic GMP (cGMP) under baseline, unstimulated conditions. Addition of ST under flow conditions increased apical and basolateral secretion of cGMP relative to the level under static conditions but did not enhance intracellular cGMP accumulation. Cyclic stretch did not have any significant effect beyond that contributed by flow. This study demonstrates that fluid flow application initiates changes in intestinal epithelial cell characteristics relative to those of static culture conditions under both baseline conditions and with exposure to ST enterotoxin and suggests that further investigations of the application of these mechanical forces will provide insights into physiology and pathophysiology that more closely resemble intact intestine than study under static conditions.

Porcine Intestinal Enteroids: a New Model for Studying Enteric Coronavirus Porcine Epidemic Diarrhea Virus Infection and the Host Innate Response.

Porcine epidemic diarrhea virus (PEDV), a member of the group of alphacoronaviruses, is the pathogen of a highly contagious gastrointestinal swine disease. The elucidation of the events associated with the intestinal epithelial response to PEDV infection has been limited by the absence of good in vitro porcine intestinal models that recapitulate the multicellular complexity of the gastrointestinal tract. Here, we generated swine enteroids from the intestinal crypt stem cells of the duodenum, jejunum, or ileum and found that the generated enteroids are able to satisfactorily recapitulate the complicated intestinal epithelium in vivo and are susceptible to infection by PEDV. PEDV infected multiple types of cells, including enterocytes, stem cells, and goblet cells, and exhibited segmental infection discrepancies compared with ileal enteroids and colonoids, and this finding was verified in vivo Moreover, the clinical isolate PEDV-JMS propagated better in ileal enteroids than the cell-adapted isolate PEDV-CV777, and PEDV infection suppressed interferon (IFN) production early during the infection course. IFN lambda elicited a potent antiviral response and inhibited PEDV in enteroids more efficiently than IFN alpha (IFN-α). Therefore, swine enteroids provide a novel in vitro model for exploring the pathogenesis of PEDV and for the in vitro study of the interplay between a host and a variety of swine enteric viruses.IMPORTANCE PEDV is a highly contagious enteric coronavirus that causes significant economic losses, and the lack of a good in vitro model system is a major roadblock to an in-depth understanding of PEDV pathogenesis. Here, we generated a porcine intestinal enteroid model for PEDV infection. Utilizing porcine intestinal enteroids, we demonstrated that PEDV infects multiple lineages of the intestinal epithelium and preferably infects ileal enteroids over colonoids and that enteroids prefer to respond to IFN lambda 1 over IFN-α. These events recapitulate the events that occur in vivo This study constitutes the first use of a primary intestinal enteroid model to investigate the susceptibility of porcine enteroids to PEDV and to determine the antiviral response following infection. Our study provides important insights into the events associated with PEDV infection of the porcine intestine and provides a valuable in vitro model for studying not only PEDV but also other swine enteric viruses.

Quantitation and chemical coding of enteroendocrine cell populations in the human jejunum.

Recent studies reveal substantial species and regional differences in enteroendocrine cell (EEC) populations, including differences in patterns of hormone coexpression, which limit extrapolation between animal models and human. In this study, jejunal samples, with no histologically identifiable pathology, from patients undergoing Whipple's procedure were investigated for the presence of gastrointestinal hormones using double- and triple-labelling immunohistochemistry and high-resolution confocal microscopy. Ten hormones (5-HT, CCK, secretin, proglucagon-derived peptides, PYY, GIP, somatostatin, neurotensin, ghrelin and motilin) were localised in EEC of the human jejunum. If only single staining is considered, the most numerous EEC were those containing 5-HT, CCK, ghrelin, GIP, motilin, secretin and proglucagon-derived peptides. All hormones had some degree of colocalisation with other hormones. This included a population of EEC in which GIP, CCK and proglucagon-derived peptides are costored, and four 5-HT cell populations, 5-HT/GIP, 5-HT/ghrelin, 5-HT/PYY, and 5-HT/secretin cell groups, and a high degree of overlap between motilin and ghrelin. The presence of 5-HT in many secretin cells is consistent across species, whereas lack of 5-HT and CCK colocalisation distinguishes human from mouse. It seems likely that the different subclasses of 5-HT cells subserve different roles. At a subcellular level, we examined the vesicular localisation of secretin and 5-HT, and found these to be separately stored. We conclude that hormone-containing cells in the human jejunum do not comply with a one-cell, one-hormone classification and that colocalisations of hormones are likely to define subtypes of EEC that have different roles.

Genomic dissection of conserved transcriptional regulation in intestinal epithelial cells.

The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs) in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS) found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from her6/HES1 that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development and physiology.

Mushroom Sparassis crispa (Hanabiratake) Fermented with Lactic Acid Bacteria Significantly Enhances Innate Immunity of Mice.

Sparassis crispa (SC; Japanese name: Hanabiratake) is a mushroom with high ß(1-3)-glucan content. We here studied the effects of SC and lactic acid bacteria-fermented SC (SCL) on innate immunity. In in vivo studies using mice, oral administration of SC or SCL enhanced the accumulation of macrophages, neutrophils, natural killer (NK) cells, and C-C chemokine receptor type 2- or phospho-Syk-expressing cells in the jejunum epithelial villi and spleen, with significantly higher cell numbers in the SCL group than in the SC group. In addition, mRNA levels of genes encoding tissue factor (TF) and tumor necrosis factor (TNF)-α were increased in monocytes/macrophages from the peritoneal cavity of mice orally administered SCL. In in vitro studies using cultured human monocytes, SC and SCL enhanced the expression of gees involved in blood coagulation and inflammation, as well as those encoding various innate immune-related factors, such as TF, TNF-α, plasminogen activator inhibitor (PAI)-1, monocyte chemotactic protein (MCP)-1, interleukin (IL)-1ß, IL-8, IL-12ß, and IL-17, in a dose-dependent manner. In particular, the expression levels of all these factors in monocytes were significantly higher with SCL treatment than with SC treatment. SCL significantly enhanced the phagocytosis of pH-sensitive fluorescent dye-labeled Escherichia coli by human monocytes compared to SC. The effect of SCL on phagocytosis was significantly reduced to approximately 30% by pre-digestion of SCL with ß-glucanase, suggesting that ß(1-3)-glucan in SCL is a major contributor to the effect. These data suggest that oral administration of SCL significantly enhances innate immunity in mice and possibly humans.

Comparative analysis of enteroendocrine cells and their hormones between mouse intestinal organoids and native tissues.

Endocrine cells in the gastrointestinal tract secrete multiple hormones to maintain homeostasis in the body. In the present study, we generated intestinal organoids from the duodenum, jejunum, and ileum of Neurogenin 3 (Ngn3)-EGFP mice and examined how enteroendocrine cells (EECs) within organoid cultures resemble native epithelial cells in the gut. Transcriptome analysis of EGFP-positive cells from Ngn3-EGFP organoids showed gene expression pattern comparable to EECs in vivo. We also compared mRNAs of five major hormones, namely, ghrelin (Ghrl), cholecystokinin (Cck), Gip, secretin (Sct), and glucagon (Gcg) in organoids and small intestine along the longitudinal axis and found that expression patterns of these hormones in organoids were similar to those in native tissues. These findings suggest that an intestinal organoid culture system can be utilized as a suitable model to study enteroendocrine cell functions in vitro.

Expression profiling of Tas2r genes reveals a complex pattern along the mouse GI tract and the presence of Tas2r131 in a subset of intestinal Paneth cells.

The chemical variability of the intestinal lumen requires the presence of molecular receptors detecting the various substances naturally occurring in the diet and as a result of the activity of the microbiota. Despite their early discovery, intestinal bitter taste receptors (Tas2r) have not yet been assigned an unambiguous physiological function. Recently, using a CRE-recombinant approach we showed that the Tas2r131 gene is expressed in a subset of mucin-producing goblet cells in the colon of mice. Moreover, we also demonstrated that the expression of the Tas2r131 locus is not restricted to this region. In the present study we aimed at characterizing the presence of positive cells also in other gastrointestinal regions. Our results show that Tas2r131+ cells appear in the jejunum and the ileum, and are absent from the stomach and the duodenum. We identified the positive cells as a subpopulation of deep-crypt Paneth cells in the ileum, strengthening the notion of a defensive role for Tas2rs in the gut. To get a broader perspective on the expression of bitter taste receptors in the alimentary canal, we quantified the expression of all 35 Tas2r genes along the gastrointestinal tract by qRT-PCR. We discovered that the number and expression level of Tas2r genes profoundly vary along the alimentary canal, with the stomach and the colon expressing the largest subsets.

Eleutheroside B increase tight junction proteins and anti-inflammatory cytokines expression in intestinal porcine jejunum epithelial cells (IPEC-J2).

Eleutheroside B (EB) is a phenylpropanoid glycoside with anti-inflammatory properties, neuroprotective abilities, immunomodulatory effects, antinociceptive effects, and regulation of blood glucose. The aim of this study was to investigate the effects of EB on the barrier function in the intestinal porcine epithelial cells J2 (IPEC-J2). The IPEC-J2 cells were inoculated into 96-well plates at a density of 5 × 103 cells per well for 100% confluence. The cells were cultured in the presence of EB at concentrations of 0, 0.05, 0.10, and 0.20 mg/ml for 48 hr. Then, 0.10 mg/ml was selected as the suitable concentration for the estimation of transepithelial electric resistance (TEER) value, alkaline phosphatase activity, proinflammatory cytokines mRNA expression, tight junction mRNA and protein expression. The results of this study indicated that the supplementation of EB in IPEC-J2 cells decreased cellular membrane permeability and mRNA expression of proinflammatory cytokines, including interleukin-6 (IL-6), interferon-γ (INF-γ), and tumour necrosis factor-α (TNF-α). The supplementation of EB in IPEC-J2 cells increased tight junction protein expression and anti-inflammatory cytokines, interleukin 10 (IL-10) and transforming growth factor beta (TGF-ß). In addition, the western blotting and real-time quantitative polymerase chain reaction (RT-qPCR) results indicated that EB significantly (p < 0.05) increased the mRNA and protein expression of intestinal tight junction proteins, Claudin-3, Occludin, and Zonula Occludins protein-1 (ZO-1). Therefore, dietary supplementation of EB may increase intestinal barrier function, tight junction protein expression, anti-inflammatory cytokines, and decrease proinflammatory cytokines synthesis in IPEC-J2 cells.