Intestinal Epithelial Cell Differentiation Research Articles

Short-chain fatty acids: linking diet, the microbiome and immunity.

The short-chain fatty acids (SCFAs) butyrate, propionate and acetate are microbial metabolites and their availability in the gut and other organs is determined by environmental factors, such as diet and use of antibiotics, that shape the diversity and metabolism of themicrobiota. SCFAs regulate epithelial barrier function as well as mucosal and systemic immunity via evolutionary conserved processes that involve G protein-coupled receptor signalling or histone deacetylase activity. Indicatively, the anti-inflammatory role of butyrate is mediated through direct effects on the differentiation of intestinal epithelial cells, phagocytes, B cells and plasma cells, and regulatory and effector T cells. Intestinally derived SCFAs also directly and indirectly affect immunity at extra-intestinal sites, such as the liver, the lungs, the reproductive tract and the brain, and have been implicated in a range of disorders, including infections, intestinal inflammation, autoimmunity, food allergies, asthma and responses to cancer therapies. An ecological understanding of microbial communities and their interrelated metabolic states, as well as the engineering of butyrogenic bacteria may support SCFA-focused interventions for the prevention and treatment of immune-mediated diseases.

Protective effects of Lactobacillus on heat stress-induced intestinal injury in finisher broilers by regulating gut microbiota and stimulating epithelial development

Heat stress (HS) is a critical challenge in broilers due to the high metabolic rate and lack of sweat glands. Results from this study show that implementing a cyclic chronic HS (34 °C for 7 h/d) to finisher broilers decreased the diversity of cecal microbiota and impaired intestinal barrier, resulting in gut leak and decreased body weight (both P < 0.05). These alterations might be related to inflammatory outbursts and the retarded proliferation of intestinal epithelial cells (IECs) according to the transcriptome analysis. Considering the potential beneficial properties of Lactobacillus on intestinal development and function, the protective effects of Lactobacillus rhamnosus (L. rhamnosus) on the intestine were investigated under HS conditions in this study. Orally supplemented L. rhamnosus improved the composition of cecal microbiota and upregulated the transcription of tight junction proteins in both duodenum and jejunum, with a consequent suppression in intestinal gene expressions of pro-inflammatory cytokines and facilitation in digestive capability. Meanwhile, the jejunal villus height of the birds that received L. rhamnosus was significantly higher compared with those treated with the broth (P < 0.05). The expression abundances of genes related to IECs proliferation and differentiation were increased by L. rhamnosus, along with upregulated mRNA levels of Wnt3a and β-catenin in jejunum. In addition, L. rhamnosus attenuated enterocyte apoptosis as indicated by decreased caspase-3 and caspase-9 gene expressions. The results indicated that oral administration with L. rhamnosus mitigated HS-induced dysfunction by promoting intestinal development and epithelial maturation in broilers and that the effects of L. rhamnosus might be dependent of Wnt/β-catenin signaling.

Potential Regulatory Gene Network Associated with the Ameliorative Effect of Oat Antibacterial Peptides on Rat Colitis.

Oat protein is unstable in intestinal fluid digestion, and it is easily degraded by trypsin and chymotrypsin, producing low molecular weight peptides. Endopeptidase hydrolysis can improve the bioavailability of active peptides and avoid further digestion in the gastrointestinal tract. Antimicrobial peptides (AMPs) can effectively improve host immunity, but most related studies focus on physiology and ecology, and there are few reports on their molecular level. Therefore, in this article, oat peptides were prepared via the simulated digestion method in vitro, and the main metabolites and action factors affecting colitis were screened by using the multi-omics methods in a high-throughput mode to analyze the effect and mechanism of colitis. Firstly, oat antimicrobial peptides were prepared from cationic resin combined with HPLC, and the anti-inflammatory effects of antimicrobial peptides were analyzed in vitro through the use of human colon epithelial (HCoEpiC) anti-inflammatory cells. In vivo experiments using rats have verified that AMPs can effectively prevent colitis caused by dextran sodium sulfate (DSS), reduce intestinal inflammatory cell infiltration and glandular disappearance in the colon, and reduce the apoptosis rate of colon cells. Secondly, metabolomics and transcriptomics were combined to analyze the mechanism of preventing enteritis, and it was found that oat antimicrobial peptides can promote DAG diglycerol production and inhibit the activation of T helper cells (TH), resulting in the down-regulation of key factors in the main downstream pathways of TH1, TH2 and TH17, and inhibit the production of inflammatory cells. At the same time, AMP can activate the wnt pathway, improve the expression of key genes of wnt and frizzled, promote the generation of intestinal stem cells, facilitate the differentiation and repair of intestinal epithelial cells, and prevent the generation of enteritis. Finally, the underlying genetic regulatory network of the important pathway was constructed from the effect of AMP on rat colitis.

Role of microencapsulated Lactobacillus plantarum in alleviating intestinal inflammatory damage through promoting epithelial proliferation and differentiation in layer chicks.

The alleviating effects of Lactobacillus plantarum in microencapsulation (LPM) on lipopolysaccharide (LPS)-induced intestinal inflammatory injury were investigated in layer chicks. A total of 252 healthy Hy-Line Brown layer chicks were randomly divided into six groups. Birds were injected with saline or LPS except for the control, and the diets of birds subjected to LPS were supplemented with nothing, L. plantarum, LPM, and wall material of LPM, respectively. The viable counts of LPM reached 109 CFU/g, and the supplemental levels of L. plantarum, LPM, and WM were 0.02 g (109 CFU), 1.0 g, and 0.98 g, per kilogram feed, respectively. LPS administration caused intestinal damage in layer chicks, evidenced by increased proinflammatory factors accompanied by poor intestinal development and morphology (p < 0.05). LPM/LPS significantly increased body weight, small intestine weight and length, villus height, villus height/crypt depth, and mRNA relative expression of tight junction protein genes (p < 0.05) and performed better than free L. plantarum. These findings could be attributed to the significant increase in viable counts of L. plantarum in the small intestine (p < 0.05), as well as the enhanced levels of Actinobacteriota, Lactobacillaceae, and Lactobacillus in intestinal microbiota (p < 0.05). Such results could further significantly increase goblet and PCNA+ cell percentage (p < 0.05); the mRNA relative expressions of epithelial cell, fast-cycling stem cell, quiescent stem cell, endocrine cell, and Paneth cell; and goblet and proliferative cell marker genes, including E-cadherin, Lgr-5, Bmi-1, ChA, Lysozome, Mucin-2, and PCNA (p < 0.05). Furthermore, the mRNA relative expressions of key genes involved in epithelial cell proliferation, namely, c-Myc, Cyclin-1, Wnt-3, Lrp-5, and Olfm-4, exhibited significant upregulation compared with the LPS treatment, as well as the differentiating genes Notch-1 and Hes-1 (p < 0.05). To sum up, microencapsulated L. plantarum supplementation could alleviate intestinal injury in layer chicks induced by LPS by promoting the proliferation and differentiation of intestinal epithelial cells, which could be attributed to the increase in viable count of L. plantarum in the gut and optimization in intestinal microbial flora.

The in vitro digestates from Brussels sprouts processed with various hydrothermal treatments affect the intestinal epithelial cell differentiation, mitochondrial polarization and glutathione level

Brussels sprouts provide bioactive compounds with widely acknowledged health-promoting effects observed in various levels: single cells, organs and tissues, or the whole organism. However, the choice of the appropriate hydrothermal processing is critical to sustain the nutritional values and cytoprotective activities, as Brussels sprouts are rarely eaten raw. The aim of this study was to evaluate the impact of various culinary methods (boiling, steaming, and sous-vide in comparison to raw plant material) applied to Brussels sprouts on the chosen functions of liver and intestinal cell lines (HepG2 and Caco-2, respectively): the markers of enterocyte differentiation (alkaline phosphatase and sucrase-isomaltase activities and protein level), glutathione store management (total GSH concentration and Glu, Gly, Cys and Met amino acids analysis) and mitochondrial polarization (JC-1 staining analysis). The in vitro digestates from raw Brussels sprouts had a stronger positive effect on the enterocyte marker enzymes in the Caco-2 cultures as compared to the digestates from the hydrothermally processed vegetables (boiled, steamed, and sous-vide). The sous-vide method diminished the intracellular glutathione stores. Hydrothermal processing, particularly steaming and sous-vide had a negative impact on the mitochondrial abundance and polarization as compared to raw vegetables. Our results suggest that shorter processing is more beneficial to retain glutathione and mitochondrial polarization than longer hydrothermal treatment.

Regulation of epithelial cell differentiation by the Ubiquitous expressed transcript isoform 1 in ulcerative colitis.

Mucosal healing has emerged as a desirable treatment goal for patients with ulcerative colitis (UC). Healing of mucosal wounds involves epithelial cell proliferation and differentiation, and Y-box transcription factor ZONAB has recently been identified as the key modulator of intestinal epithelial restitution. We studied the characteristics of UXT-V1 expression in UC patients using immunohistochemistry and qPCR. The functional role of UXT-V1 in the colonic epithelium was investigated using lentivirus-mediated shRNA in vitro and ex vivo. Through endogenous Co-immunoprecipitation and LC-MS/MS, we identified ZONAB as a UXT-V1-interactive protein. Herein, we report that UXT-V1 promotes differentiation of intestinal epithelial cells by regulating the nuclear translocation of ZONAB. UXT-V1 was upregulated in the intestinal epithelia of UC patients compared with that of healthy controls. Knocking down UXT-V1 in NCM-460 cells led to the enrichment of pathways associated with proliferation and differentiation. Furthermore, the absence of UXT-V1 in cultured intestinal epithelial cells and colonic organoids inhibited differentiation to the goblet cell phenotype. Mechanistically, the loss of UXT-V1 in the intestinal epithelial cells allowed nuclear translocation of ZONAB, wherein it regulated the transcription of differentiation-related genes, including AML1 and KLF4. Taken together, our study reveals a potential role of UXT-V1 in regulating epithelial cell differentiation, proving a molecular basis for mucosal healing in UC.

Mettl14-mediated m6 A modification regulates the abnormal differentiation of small intestinal epithelial stem cells in diabetic state.

Diabetes mellitus (DM) and its related complications are a global epidemic characterized by high morbidity and mortality. However, little is known about diabetic enteropathy (DE) and its the potential underlying mechanism. Intestinal epithelial stem cells (IESCs) were harvested from experimental mice, and the levels of dominant N6-methyladenosine (m6 A)-related enzyme were detected by RT-PCR, Western blotting, immunohistochemistry. The role of Mettl14 in the abnormal differentiation of intestinal epithelial cells (IECs) during DM was confirmed by knockdown experiments. RT-PCR, MeRIP, and bioinformatics analysis were carried out to confirm the downstream target of Mettl14. Through bioinformatics analysis, RT-PCR, and Western blotting, we further analyzed the differentiation-related gene in the IECs from mice with DM. In this study, the levels of Mettl14 and m6 A were higher in db/db mice than that in control mice. And abnormal differentiation of IECs in DM was associated with Mettl14 overexpression. Additionally, Mettl14 is a major determinant of IESCs identity and organoid-forming upon DM state. Mechanistically, we revealed that the candidate binding target of Mettl14 was Fzd2 mRNA and affected Fzd2 stability. Moreover, Mettl14 downregulation was observed to attenuate the abnormal differentiation of IECs through modulating Fzd2 m6A modification in DM state. Together, our results provide definitive evidence for the essential role of Mettl14 in differentiation of IESCs in DM state.

Integrin α7β1 represses intestinal absorptive cell differentiation

Intestinal epithelial cell differentiation is a highly controlled and orderly process occurring in the crypt so that cells migrating out to cover the villi are already fully functional. Absorptive cell precursors, which originate from the stem cell population located in the lower third of the crypt, are subject to several cycles of amplification in the transit amplifying (TA) zone, before reaching the terminal differentiation compartment located in the upper third. There is a large body of evidence that absorptive cell differentiation is halted in the TA zone through various epigenetic, transcriptional and intracellular signalling events or mechanisms allowing the transient expansion of this cell population but how these mechanisms are themself regulated remains obscure. One clue can be found in the epithelial cell-matrix microenvironment located all along the crypt-villus axis. Indeed, a previous study from our group revealed that α5-subunit containing laminins such as lamimin-511 and 512 inhibit early stages of differentiation in Caco-2/15 cells. Among potential receptors for laminin 511/512 is the integrin α7β1, which has previously been reported to be expressed in the human intestinal crypts and in early stages of Caco-2/15 cell differentiation. In this study, the effects of knocking down ITGA7 in Caco-2/15 cells were studied using shRNA and CRISPR/Cas9 strategies. Abolition of the α7 integrin subunit resulted in a significant increase in the level of differentiation and polarization markers as well as the morphological features of intestinal cells. Activities of focal adhesion kinase and Src kinase were both reduced in α7-knockdown cells while the three major intestinal pro-differentiation factors CDX2, HNFα1 and HNF4α were overexpressed. Two epigenetic events associated with intestinal differentiation, the reduction of tri-methylated lysine 27 on histone H3 and the increase of acetylation of histone H4 were also observed in α7-knockdown cells. On the other hand, the ablation of α7 had no effect on cell proliferation. In conclusion, these data indicate that integrin α7β1 acts as a major repressor of absorptive cell terminal differentiation in the Caco-2/15 cell model and suggest that the laminin-α7β1 integrin interaction occurring in the transit amplifying zone of the adult intestine is involved in the transient halting of absorptive cell terminal differentiation.

Vitronectin Destroyed Intestinal Epithelial Cell Differentiation through Activation of PDE4-Mediated Ferroptosis in Inflammatory Bowel Disease.

Vitronectin (VTN) has been reported to trigger cell pyroptosis to aggravate inflammation in our previous study. However, the function of VTN in inflammatory bowel disease (IBD) remains to be addressed. Real-time PCR and western blotting were performed to analyze VTN-regulated intestinal epithelial cell (IEC) differentiation through ferroptosis, and immunofluorescence (IF), luciferase, and chromatin immunoprecipitation were used to identify whether VTN-modulated ferroptosis is dependent on phosphodiesterase 4 (PDE4)/protein kinase A (PKA)/cyclic adenosine monophosphate-response element-binding protein (CREB) cascade pathway. In vivo experiment in mice and a pilot study in patients with IBD were used to confirm inhibition of PDE4-alleviated IECs ferroptosis, leading to cell differentiation during mucosal healing. Herein, we found that caudal-related homeobox transcription factor 2-mediated IECs differentiation was impaired in response to VTN, which was attributed to enhanced ferroptosis characterized by decreased glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 expression. Inhibition of ferroptosis in IECs rescued the inhibitory effect of VTN on cell differentiation. Further analysis showed that VTN triggered phosphorylation of PDE4, leading to inhibit PKA/CREB activation and CREB nuclear translocation, which further reduced GPX4 transactivation. Endogenous PKA interacted with CREB, and this interaction was destroyed in response to VTN stimulation. What is more, overexpression of CREB in CaCO2 cells overcame the promotion of VTN on ferroptosis. Most importantly, inhibition of PDE4 by roflumilast or dipyridamole could alleviate dextran sulfate sodium-induced colitis in mice and in a pilot clinical study confirmed by IF. These findings demonstrated that highly expressed VTN disrupted IECs differentiation through PDE4-mediated ferroptosis in IBD, suggesting targeting PDE4 could be a promising therapeutic strategy for patients with IBD.

Alleviative Effect of Probiotic Ferment on Lawsonia intracellularis Infection in Piglets.

(1) Background: Lawsonia intracellularis (LI) is an obligate intracellular Gram-negative bacterium that causes porcine ileitis. Pigs infected with LI have severe ileal lesions and show symptoms of diarrhea, indigestion, and growth retardation. Previous studies found that probiotic ferment (FAM) improved the growth performance, gut barrier, and function in piglets. Therefore, we aimed to reveal the mechanism that FAM alleviates negative performance in LI-challenged piglets by characterizing the changes in intestinal integrity, function, and gut microbiota following FAM supplementation. (2) Methods: Twenty-four healthy piglets were randomly allotted to four treatments. Three groups were challenged with LI; both FAM addition and vaccination were performed to explore their positive effects on LI-infected piglets. (3) Results: Piglets infected with LI showed lower growth performance and typical pathological symptoms. Moreover, microscopic images showed that observed intestinal morphological damage could be repaired by FAM and vaccine. To explore the digestion of nutrients in piglets, both digestive enzyme activity and ileal transporter expression were performed to reveal the promoting effect of additives. Reduction of LI colonization intervention by FAM could also ameliorate abnormal differentiation and function of intestinal epithelial cells and alleviate severe inflammatory responses in piglets. Regarding the gut microbiota, both the structure and function of the ileal and colonic microbiota were altered following FAM supplementation. (4) Conclusions: In conclusion, probiotic ferment can reduce the colonization of LI in the ileum, improve intestinal damage, barrier function and microbiota structure, and enhance digestive enzyme activity and nutrient transport proteins expression, thereby improving piglet growth performance, which has the effect of preventing ileitis in pigs.

Epigenetic mechanisms of epithelial – immune interactions that shape immune surveillance in the intestinal epithelium

Abstract Immune surveillance of the intestinal epithelium is regulated by the reciprocal interactions between intestinal epithelial cells, immune system and microbiome. This highly specialized compartment maintains the tolerance against dietary products and commensal bacteria while protecting the epithelium against pathogens, cancer risk and tissue damage. While recent studies identified epigenetic mechanisms that orchestrate the self-renewal and differentiation of intestinal epithelial cells (IECs), little is known about the mechanisms that govern epithelial – immune interactions and how they influence immune surveillance and tissue regeneration. Here we dissect epithelial-intrinsic epigenetic features including transcription factors and co-activators that govern immune surveillance of intestinal epithelium using genetically engineered mouse models and autologous human patient-derived organoid – immune co-cultures. Ablation of epigenetic regulators that associate with immune interaction gene modules specifically in IECs dampens intraepithelial lymphocyte-mediated immune surveillance. We identified precise epigenetic mechanisms of epithelial – immune interactions including promoter and enhancer regulation by transcription factors and co-activators both in mice and humans. Finally, disruption of the epigenetic regulators that maintain epithelial – immune interactions lead to impaired responses in clinically-relevant mouse models of infection, tissue regeneration and cancer. These results establish a strong foundation towards understanding the epigenetic mechanisms that govern multi-cellular interactions within the barrier tissues in physiology or disease states. Supported by grants from Mathers Foundation, STARR Cancer Consortium (I13-0052), The Mark Foundation for Cancer Research (20-028-EDV), NIH (P30CA045508-33), CZI Ancestry Network for the Human Cell Atlas

Amniotic fluid stem cell attenuated necrotizing enterocolitis progression by promoting Rspo3/AMPKα axis

R-spondin 3 (Rspo3) is involved in various cellular processes. The alteration of Rspo3 participates in the differentiation of intestinal epithelial cells which are the crucial effector cells during necrotizing enterocolitis (NEC) development. Amniotic fluid stem cells (AFSCs) were recently indicated as a potential approach for NEC therapy. This study aimed to illustrate the regulatory role and mechanism of Rspo3 in the pathogenesis of NEC and whether AFSCs therapy would impact NEC by mediating Rspo3. First, the alteration of Rspo3 was investigated in the serum and tissues of NEC patients, and an in vitro cell model induced by LPS. A gain-of-function assay was conducted to explore the function of Rspo3 in NEC. Through the analysis of adenosine 5‘-monophosphate-activated protein kinase α (AMPKα) activation, the mechanism of Rspo3-mediated NEC progression was demonstrated. Finally, AFSCs were used to coculture human intestinal epithelial cells (HIECs) and the impacts on NEC development were also explored. The results found that Rspo3 was dramatically depressed during NEC progression and reversing Rspo3 expression ameliorated LPS-induced injury, inflammation, oxidative stress and tight junction dysregulation in HIECs. Besides, Rspo3 overexpression reversed AMPKα inactivation induced by NEC and an AMPKα inhibitor, Compound C, blocked the effect of Rspo3 overexpression on NEC. AFSCs treatment was beneficial for NEC therapy by restoring Rspo3 expression which was counteracted by exosome inhibitor. Generally, AFSCs attenuated NEC progression by promoting the Rspo3/AMPKα axis which might exert via the secretion of exosomes. Our conclusions might be valuable for NEC diagnosis and therapy.

IPA and its precursors differently modulate the proliferation, differentiation, and integrity of intestinal epithelial cells.

Indole-3-propionic acid (IPA) is a tryptophan-derived microbial metabolite that has been associated with protective effects against inflammatory and metabolic diseases. However, there is a lack of knowledge regarding the effects of IPA under physiological conditions and at the intestinal level. Human intestinal epithelial Caco-2 cells were treated for 2, 24, and/or 72 h with IPA or its precursors - indole, tryptophan, and propionate - at 1, 10, 100, 250, or 500 μM to assess cell viability, integrity, differentiation, and proliferation. IPA induced cell proliferation and this effect was associated with a higher expression of extracellular signal-regulated kinase 2 (ERK2) and a lower expression of c-Jun. Although indole and propionate also induced cell proliferation, this involved ERK2 and c-Jun independent mechanisms. On the other hand, both tryptophan and propionate increased cell integrity and reduced the expression of claudin-1, whereas propionate decreased cell differentiation. In conclusion, these findings suggested that IPA and its precursors distinctly contribute to the proliferation, differentiation, and barrier function properties of human intestinal epithelial cells. Moreover, the pro-proliferative effect of IPA in intestinal epithelial cells was not explained by its precursors and is rather related to its whole chemical structure. Maintaining IPA at physiological levels, e.g., through IPA-producing commensal bacteria, may be important to preserve the integrity of the intestinal barrier and play an integral role in maintaining metabolic homeostasis.

The lncRNA Tincr Regulates the Abnormal Differentiation of Intestinal Epithelial Stem Cells in the Diabetic State Via the miR-668-3p/Klf3 Axis.

Diabetes mellitus (DM) is among the most common chronic diseases, and diabetic enteropathy (DE), which is a complication caused by DM, is a serious health condition. Long noncoding RNAs (lncRNAs) are regulators of DE progression. However, the mechanisms of action of multiple lncRNAs involved in DE remain poorly understood. Reverse transcription-quantitative PCR (RT-qPCR) and in situ hybridization were used to analyze terminal differentiation-induced lncRNA (Tincr) expression in intestinal epithelial cells (IECs) in the DM state. Microarray analysis, bioinformatics analysis, and luciferase reporter assays were used to identify the genes targeted by Tincr. The role of miR-668-3p was then explored by up- and down-regulating its expression in vitro and in vivo. In this study, we observed that the level of lncRNA Tincr was increased in IECs in the DM state. More importantly, Tincr was associated with abnormal intestinal epithelial stem cell (IESC) differentiation in DM. Our mechanistic study demonstrated that Tincr is a major marker of Lgr5+ stem cells in DM. In addition, we investigated whether Tincr directly targets miR-668-3p and whether miR-668-3p targets Klf3. Our findings showed that Tincr sponged miR-668-3p, which attenuated abnormal IESC differentiation in DM by regulating Klf3 expression. This study presents evidence of an essential role for Tincr in IESC differentiation in DM.

Drinking alkaline mineral water confers diarrhea resistance in maternally separated piglets by maintaining intestinal epithelial regeneration via the brain-microbe-gut axis

IntroductionDiarrhea has the fourth-highest mortality rate of all diseases and causes a large number of infant deaths each year. The maternally separated (MS) piglet (newly weaned piglet) is an excellent model to investigate the treatment of diarrhea in infants. Drinking alkaline mineral water has the potential to be therapeutic in gastrointestinal disorders, particularly diarrhea, but the supporting evidence from system studies and the mechanisms involved have yet to be reported. ObjectivesThis study aims to determine whether drinking alkaline mineral water confers diarrhea resistance in MS piglets under weaning stress and what the fundamental mechanisms involved are. MethodsMS piglets were used to create a stress-induced intestinal disorder-diarrhea susceptibility model. A total of 240 MS piglets were randomly divided into two groups (6 pens/group and 20 piglets/pen). IPEC-J2 cell line was used for in vitro evaluation. An alkaline mineral complex (AMC) water was employed, and its effect on the hypothalamus-pituitary-adrenocortical (HPA) axis, gut microbes, gut morphology, and intestinal epithelial cell (IEC) proliferation and differentiation were investigated using a variety of experimental methodology. ResultsAMC water reduced diarrhea rate in MS piglets by inhibiting the HPA axis, ameliorating gut microbiota structure, and stimulating IEC proliferation and differentiation. Apparently, the brain-microbe-gut axis is linked with AMC water conferring diarrhea resistance in piglets. Mechanistically, AMC water decreased stress hormones (COR and Hpt) secretion by suppressing HPA axis, which then increased the abundance of beneficial gut microbes; accordingly, maintained the proliferation of IEC and promoted the differentiation of intestinal stem cells (ISC) into goblet cell and Paneth cell by activating the Wnt/β-catenin signaling pathway. In the absence of gut microbiota (in vitro), AMC activated the LPS-induced Wnt/β-catenin signaling inhibition in IPEC-J2 cells and significantly increased the number of Lgr5 + cells, whereas had no effect on IPEC-J2 differentiation. ConclusionDrinking alkaline mineral water confers diarrhea resistance in MS piglets by maintaining intestinal epithelial regeneration via the brain-microbe-gut axis; thus, this study provides a potential prevention strategy for young mammals at risk of diarrhea.

HNF4α Acts as Upstream Functional Regulator of Intestinal Wnt3 and Paneth Cell Fate

The intestinal epithelium intrinsically renews itself exvivo via the proliferation of Lgr5+ intestinal stem cells, which is sustained by the establishment of an epithelial stem cell niche. Differentiated Paneth cells are the main source of epithelial-derived niche factor supplies and produce Wnt3 as an essential factor in supporting Lgr5+ stem cell activity in the absence of redundant mesenchymal Wnts. Maturation of Paneth cells depends on canonical Wnt signaling, but few transcriptional regulators have been identified to this end. The role of HNF4α in intestinal epithelial cell differentiation is considered redundant with its paralog HNF4γ. However, it is unclear whether HNF4α alone controls intrinsic intestinal epithelial cell growth and fate in the absence of a mesenchymal niche. We used transcriptomic analyses to dissect the role of HNF4α in the maintenance of jejunal epithelial culture when cultured exvivo as enteroids in the presence or absence of compensatory mesenchymal cells. HNF4α plays a crucial role in supporting the growth and survival of jejunal enteroids. Transcriptomic analyses revealed an autonomous function of HNF4α in Wnt3 transcriptional regulation and Paneth cell differentiation. We showed that Wnt3a supplementation or co-culture with intestinal subepithelial mesenchymal cells reversed cell death and transcriptional changes caused by the deletion of Hnf4a in jejunal enteroids. Our results support the intrinsic epithelial role of HNF4α in regulating Paneth cell homeostasis and intestinal epithelium renewal in the absence of compensatory Wnt signaling.

PGC-1α in mediating mitochondrial biogenesis and intestinal epithelial differentiation promoted by purple potato extract

• Purple potato polyphenolic extract (PPE) promotes gut epithelial differentiation. • PPE upregulates PGC-1α and enhances mitochondria biogenesis in Caco-2 cells. • PPE promotes intracellular oxidative metabolism. • PGC-1α is indispensable in PPE-induced differentiation & mitochondrial biogenesis. Intestinal epithelial differentiation plays an important role in maintaining gut health and is impaired due to many diseases. Purple potatoes are rich in polyphenols that promote the differentiation of intestinal epithelial cells. In this study, we found that purple potato (PP) extract up-regulated the level of peroxisome proliferator-activated receptor γ coactivator (PGC)-1α, the expression of mitochondrial-related enzymes, the content of mitochondrial DNA, and the levels of tricarboxylic acid cycle metabolites in human intestinal epithelial Caco-2 cells. Inhibition of PGC-1α by either PGC-1α inhibitor SR-18292 or siRNA-mediated knockdown eliminated the enhancing effects of PP extract on the expression of differentiation markers, mitochondrial metabolite contents and mitochondrial DNA copy number, as well as the expression of mitochondrial DNA-encoded enzymes. In summary, PP extract promoted mitochondrial biogenesis and the differentiation of intestinal epithelial cells, and these promotion effects are dependent on the activation of PGC-1α.

Myosin Vb as a tumor suppressor gene in intestinal cancer.

Colorectal cancer causes >900,000 deaths every year and a deeper understanding of the molecular mechanisms underlying this disease will contribute to improve its clinical management and survival. Myosin Vb (MYO5B) regulates intracellular vesicle trafficking, and inactivation of this myosin disrupts the polarization and differentiation of intestinal epithelial cells causing microvillous inclusion disease (MVID), a rare congenital disorder characterized by intractable life-threatening diarrhea. Here, we show that the loss Myosin Vb interfered with the differentiation/polarization of colorectal cancer cells. Although modulation of Myosin Vb expression did not affect the proliferation of colon cancer cells, MYO5B inactivation increased their migration, invasion, and metastatic potential. Moreover, Myo5b inactivation in an intestine-specific knockout mouse model caused a >15-fold increase in the number of azoxymethane-initiated small intestinal tumors. Consistently, reduced expression of Myosin Vb in a cohort of 155 primary colorectal tumors was associated with shorter patient survival. In conclusion, we show here that loss of Myosin Vb reduces polarization/differentiation of colon cancer cells while enhancing their metastatic potential, demonstrating a tumor suppressor function for this myosin. Moreover, reduced expression of Myosin Vb in primary tumors identifies a subset of poor prognosis colorectal cancer patients that could benefit from more aggressive therapeutic regimens.

Intestinal Epithelial STAT6 Activation Rescues the Defective Anti-Helminth Responses Caused by Ogt Deletion.

Dynamic regulation of intestinal epithelial cell (IEC) proliferation and differentiation is crucial for maintaining mucosa homeostasis and the response to helminth infection. O-GlcNAc transferase (OGT), an enzyme catalyzing the transfer of GlcNAc from the donor substrate UDP-GlcNAc onto acceptor proteins, has been proposed to promote intestinal epithelial remodeling for helminth expulsion by modifying and activating epithelial STAT6, but whether the IEC intrinsic OGT-STAT6 axis is involved in anti-helminth responses has not been tested in vivo. Here, we show that the inducible deletion of Ogt in IECs of adult mice leads to reduced tuft and goblet cell differentiation, increased crypt cell proliferation, and aberrant Paneth cell localization. By using a mouse model with concurrent Ogt deletion and STAT6 overexpression in IECs, we provide direct in vivo evidence that STAT6 acts downstream of OGT to control tuft and goblet cell differentiation in IECs. However, epithelial OGT regulates crypt cell proliferation and Paneth cell differentiation in a STAT6-independent pathway. Our results verify that protein O-GlcNAcylation in IECs is crucial for maintaining epithelial homeostasis and anti-helminthic type 2 immune responses.

The miR-181 family regulates colonic inflammation through its activity in the intestinal epithelium.

The intestinal epithelium is a key physical interface that integrates dietary and microbial signals to regulate nutrient uptake and mucosal immune cell function. The transcriptional programs that regulate intestinal epithelial cell (IEC) quiescence, proliferation, and differentiation have been well characterized. However, how gene expression networks critical for IECs are posttranscriptionally regulated during homeostasis or inflammatory disease remains poorly understood. Herein, we show that a conserved family of microRNAs, miR-181, is significantly downregulated in IECs from patients with inflammatory bowel disease and mice with chemical-induced colitis. Strikingly, we showed that miR-181 expression within IECs, but not the hematopoietic system, is required for protection against severe colonic inflammation in response to epithelial injury in mice. Mechanistically, we showed that miR-181 expression increases the proliferative capacity of IECs, likely through the regulation of Wnt signaling, independently of the gut microbiota composition. As epithelial reconstitution is crucial to restore intestinal homeostasis after injury, the miR-181 family represents a potential therapeutic target against severe intestinal inflammation.