ABSTRACT
Tissues such as the intestine harbor stem cells that have remarkable functional plasticity in response to a dynamic environment. To adapt to the environment, stem cells constantly receive information from their surrounding microenvironment (also called the 'niche') that instructs them how to adapt to changes. The Drosophila midgut shows morphological and functional similarities to the mammalian small intestine and has been a useful model system to study signaling events in stem cells and tissue homeostasis. In this review, we summarize the current understanding of the Drosophila midgut regarding how stem cells communicate with microenvironmental niches including enteroblasts, enterocytes, enteroendocrine cells and visceral muscles to coordinate tissue regeneration and homeostasis. In addition, distant cells such as hemocytes or tracheal cells have been shown to interact with stem cells and influence the development of intestinal diseases. We discuss the contribution of stem cell niches in driving or counteracting disease progression, and review conceptual advances derived from the Drosophila intestine as a model for stem cell biology.
Subject(s)
Enterocytes , Intestines , Animals , Enterocytes/physiology , Stem Cells , Drosophila/physiology , Homeostasis , MammalsABSTRACT
BACKGROUND & AIMS: Excessive shedding of apoptotic enterocytes into the intestinal lumen is observed in inflammatory bowel disease and is correlated with disease relapse. Based on their cytolytic capacity and surveillance behavior, we investigated whether intraepithelial lymphocytes expressing the γδ T cell receptor (γδ IELs) are actively involved in the shedding of enterocytes into the lumen. METHODS: Intravital microscopy was performed on GFP γδ T cell reporter mice treated with intraperitoneal lipopolysaccharide (10 mg/kg) for 90 minutes to induce tumor necrosis factor-mediated apoptosis. Cell shedding in various knockout or transgenic mice in the presence or absence of blocking antibody was quantified by immunostaining for ZO-1 funnels and cleaved caspase-3 (CC3). Granzyme A and granzyme B release from ex vivo-stimulated γδ IELs was quantified by enzyme-linked immunosorbent assay. Immunostaining for γδ T cell receptor and CC3 was performed on duodenal and ileal biopsies from controls and patients with Crohn's disease. RESULTS: Intravital microscopy of lipopolysaccharide-treated mice revealed that γδ IELs make extended contact with shedding enterocytes. These prolonged interactions require CD103 engagement by E-cadherin, and CD103 knockout or blockade significantly reduced lipopolysaccharide-induced shedding. Furthermore, we found that granzymes A and B, but not perforin, are required for cell shedding. These extracellular granzymes are released by γδ IELs both constitutively and after CD103/E-cadherin ligation. Moreover, we found that the frequency of γδ IEL localization to CC3-positive enterocytes is increased in Crohn's disease biopsies compared with healthy controls. CONCLUSIONS: Our results uncover a previously unrecognized role for γδ IELs in facilitating tumor necrosis factor-mediated shedding of apoptotic enterocytes via CD103-mediated extracellular granzyme release.
Subject(s)
Antigens, CD/metabolism , Crohn Disease/metabolism , Enterocytes/physiology , Granzymes/metabolism , Integrin alpha Chains/metabolism , Intraepithelial Lymphocytes/physiology , Adolescent , Adult , Animals , Antigens, CD/genetics , Apoptosis , Cadherins/metabolism , Caspase 3/metabolism , Crohn Disease/pathology , Duodenum/pathology , Enterocytes/metabolism , Female , Gene Knockdown Techniques , Humans , Ileum/pathology , Integrin alpha Chains/genetics , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Intraepithelial Lymphocytes/enzymology , Intraepithelial Lymphocytes/pathology , Intravital Microscopy , Jejunum/immunology , Jejunum/pathology , Lipopolysaccharides , Male , Mice , Mice, Knockout , Middle Aged , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Tumor Necrosis Factor-alpha/metabolism , Young AdultABSTRACT
Enterocytes are specialized epithelial cells lining the luminal surface of the small intestine that build densely packed arrays of microvilli known as brush borders. These microvilli drive nutrient absorption and are arranged in a hexagonal pattern maintained by intermicrovillar links formed by 2 nonclassical members of the cadherin superfamily of calcium-dependent cell adhesion proteins: protocadherin-24 (PCDH24, also known as CDHR2) and the mucin-like protocadherin (CDHR5). The extracellular domains of these proteins are involved in heterophilic and homophilic interactions important for intermicrovillar function, yet the structural determinants of these interactions remain unresolved. Here, we present X-ray crystal structures of the PCDH24 and CDHR5 extracellular tips and analyze their species-specific features relevant for adhesive interactions. In parallel, we use binding assays to identify the PCDH24 and CDHR5 domains involved in both heterophilic and homophilic adhesion for human and mouse proteins. Our results suggest that homophilic and heterophilic interactions involving PCDH24 and CDHR5 are species dependent with unique and distinct minimal adhesive units.
Subject(s)
Cadherin Related Proteins/ultrastructure , Microvilli/pathology , Animals , Caco-2 Cells , Cadherin Related Proteins/metabolism , Cadherins/metabolism , Carrier Proteins/metabolism , Cell Adhesion , Cell Adhesion Molecules/metabolism , Cell Communication , Cell Line , Enterocytes/metabolism , Enterocytes/physiology , Epithelial Cells/metabolism , Humans , Intestine, Small/pathology , Intestine, Small/physiology , Mice , Microvilli/physiology , Species SpecificityABSTRACT
Hypoxia and hyperthermia, which can be induced by high environmental temperature or strenuous exercise, are two common stressors that affect intestinal epithelial integrity and lead to multiple clinical symptoms. In this study, we developed an in-vitro intestinal monolayer model using two human colonic epithelial cell lines, Caco-2 and HT-29, co-cultured in Transwell inserts, and investigated the effects of heat treatment and/or hypoxia on the epithelial barrier function. The monolayer with a ratio of 9:1 (Caco-2:HT-29) showed high trans-epithelial electrical resistance (TEER), low Lucifer Yellow permeability and high mucin production. Hyperthermia and/or hypoxia exposure (2 h) triggered heat shock and oxidative stress responses. HSP-70 and HSF-1 protein levels were up-regulated by hyperthermia, which were further enhanced when hyperthermia was combined with hypoxia. Increased HIF-1α protein expression and Nrf2 nuclear translocation was only caused by hypoxia. Hyperthermia and/or hypoxia exposure disrupted the established monolayer by increasing paracellular permeability, decreasing ZO-1, claudin-3 and occludin protein/mRNA expression, while enhancing E-cadherin protein expression. Tight junction protein distribution in the monolayer was also modulated by the hyperthermia and/or hypoxia exposure. In addition, transcription levels of mucin genes, MUC-2 and MUC-5AC, were increased after 2 h of hyperthermia and/or hypoxia exposure. In conclusion, this Caco-2/HT-29 cell model is valid and effective for studying detrimental effects of hyperthermia and/or hypoxia on intestinal barrier function and related heat shock and oxidative stress pathways and can be used to investigate possible interventions to reverse hyperthermia and/or hypoxia-induced intestinal epithelial injury.
Subject(s)
Cell Hypoxia , Enterocytes/physiology , Goblet Cells/physiology , Heat-Shock Response , Adenocarcinoma/pathology , Adenocarcinoma, Mucinous/pathology , Cell Hypoxia/genetics , Cell Hypoxia/physiology , Cell Line, Tumor , Coculture Techniques , Colonic Neoplasms/pathology , Coloring Agents , Electric Impedance , Gene Expression Regulation, Neoplastic , Heat-Shock Response/genetics , Heat-Shock Response/physiology , Humans , Intercellular Junctions , Isoquinolines , Mucins/biosynthesis , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Oxidative Stress , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , Transcription, GeneticABSTRACT
SCOPE: Human milk can prevent the development of necrotizing enterocolitis (NEC). Human milk is rich in cargo-carrying exosomes that participate in intercellular communication. This study investigated the effects of term and preterm human milk-derived exosomes, and elucidated their lipid expression profiles. METHODS AND RESULTS: Milk from healthy mothers is collected who have delivered full-term or preterm infants, and exosomes are isolated and quantified. Administration of term and preterm milk exosomes significantly enhances epithelial proliferation and migration in vitro, and ameliorates the severity of NEC in vivo. A total of 395 lipids are identified in term and preterm human milk-derived exosomes. Bioinformatics analysis and western blotting reveal that top 50 lipids regulate intestinal epithelial cell function via the Extracellular-Signal-Regulated Kinase/Mitogen Activated Protein Kinase (ERK/MAPK) pathway. CONCLUSION: This study reveals for the first time the lipidomic complexities in exosomes derived from preterm and term milk. The results provide novel mechanistic insight on how human milk prevents the development of NEC.
Subject(s)
Exosomes/chemistry , Exosomes/physiology , Lipids/analysis , Lipids/physiology , Milk, Human/cytology , Animals , Animals, Newborn , Cell Proliferation , Enterocolitis, Necrotizing/prevention & control , Enterocytes/drug effects , Enterocytes/physiology , Exosomes/ultrastructure , Female , Humans , Infant, Newborn , Infant, Premature , Lipopolysaccharides/pharmacology , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Microscopy, Electron , RatsABSTRACT
The intestine is a site of direct encounter with the external environment and must consequently balance barrier defense with nutrient uptake. To investigate how nutrient uptake is regulated in the small intestine, we tested the effect of diets with different macronutrient compositions on epithelial gene expression. We found that enzymes and transporters required for carbohydrate digestion and absorption were regulated by carbohydrate availability. The "on-demand" induction of this machinery required γδ T cells, which regulated this program through the suppression of interleukin-22 production by type 3 innate lymphoid cells. Nutrient availability altered the tissue localization and transcriptome of γδ T cells. Additionally, transcriptional responses to diet involved cellular remodeling of the epithelial compartment. Thus, this work identifies a role for γδ T cells in nutrient sensing.
Subject(s)
Dietary Carbohydrates/administration & dosage , Dietary Carbohydrates/metabolism , Enterocytes/physiology , Interleukins/metabolism , Intestinal Mucosa/physiology , Receptors, Antigen, T-Cell, gamma-delta , T-Lymphocyte Subsets/physiology , Adaptation, Physiological , Animals , Cell Communication , Dietary Proteins/administration & dosage , Digestion , Gene Expression Regulation , Interleukins/genetics , Intestinal Absorption , Intestinal Mucosa/cytology , Intestine, Small/cytology , Intestine, Small/metabolism , Mice, Inbred C57BL , Nutrients/administration & dosage , Nutrients/metabolism , T-Lymphocyte Subsets/immunology , Transcription, Genetic , Transcriptome , Interleukin-22ABSTRACT
A hallmark of aging is loss of differentiated cell identity. Aged Drosophila midgut differentiated enterocytes (ECs) lose their identity, impairing tissue homeostasis. To discover identity regulators, we performed an RNAi screen targeting ubiquitin-related genes in ECs. Seventeen genes were identified, including the deubiquitinase Non-stop (CG4166). Lineage tracing established that acute loss of Non-stop in young ECs phenocopies aged ECs at cellular and tissue levels. Proteomic analysis unveiled that Non-stop maintains identity as part of a Non-stop identity complex (NIC) containing E(y)2, Sgf11, Cp190, (Mod) mdg4, and Nup98. Non-stop ensured chromatin accessibility, maintaining the EC-gene signature, and protected NIC subunit stability. Upon aging, the levels of Non-stop and NIC subunits declined, distorting the unique organization of the EC nucleus. Maintaining youthful levels of Non-stop in wildtype aged ECs safeguards NIC subunits, nuclear organization, and suppressed aging phenotypes. Thus, Non-stop and NIC, supervise EC identity and protects from premature aging.
Subject(s)
Aging, Premature/genetics , Aging/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/physiology , Enterocytes/physiology , Animals , Disease Models, Animal , Drosophila Proteins/metabolism , Female , Male , Phenotype , ProteomeABSTRACT
BACKGROUND AND AIMS: Intestinal epithelial cells [IECs] from inflammatory bowel disease [IBD] patients exhibit an excessive induction of endoplasmic reticulum stress [ER stress] linked to altered intestinal barrier function and inflammation. Colonic tissues and the luminal content of IBD patients are also characterized by increased serine protease activity. The possible link between ER stress and serine protease activity in colitis-associated epithelial dysfunctions is unknown. We aimed to study the association between ER stress and serine protease activity in enterocytes and its impact on intestinal functions. METHODS: The impact of ER stress induced by Thapsigargin on serine protease secretion was studied using either human intestinal cell lines or organoids. Moreover, treating human intestinal cells with protease-activated receptor antagonists allowed us to investigate ER stress-resulting molecular mechanisms that induce proteolytic activity and alter intestinal epithelial cell biology. RESULTS: Colonic biopsies from IBD patients exhibited increased epithelial trypsin-like activity associated with elevated ER stress. Induction of ER stress in human intestinal epithelial cells displayed enhanced apical trypsin-like activity. ER stress-induced increased trypsin activity destabilized intestinal barrier function by increasing permeability and by controlling inflammatory mediators such as C-X-C chemokine ligand 8 [CXCL8]. The deleterious impact of ER stress-associated trypsin activity was specifically dependent on the activation of protease-activated receptors 2 and 4. CONCLUSIONS: Excessive ER stress in IECs caused an increased release of trypsin activity that, in turn, altered intestinal barrier function, promoting the development of inflammatory process.
Subject(s)
Colitis, Ulcerative/pathology , Crohn Disease/pathology , Endoplasmic Reticulum Stress/physiology , Enterocytes/physiology , Intestinal Absorption/physiology , Trypsin/metabolism , Cell Culture Techniques , Cell Line , Colitis, Ulcerative/etiology , Colitis, Ulcerative/metabolism , Crohn Disease/etiology , Crohn Disease/metabolism , Humans , Organoids , ThapsigarginABSTRACT
BACKGROUND & AIMS: Notch signaling coordinates cell differentiation processes in the intestinal epithelium. The transcription factor Nrf2 orchestrates defense mechanisms by regulating cellular redox homeostasis, which, as shown previously in murine liver, can be amplified through signaling crosstalk with the Notch pathway. However, interplay between these 2 signaling pathways in the gut is unknown. METHODS: Mice modified genetically to amplify Nrf2 in the intestinal epithelium (Keap1f/f::VilCre) were generated as well as pharmacological activation of Nrf2 and subjected to phenotypic and cell lineage analyses. Cell lines were used for reporter gene assays together with Nrf2 overexpression to study transcriptional regulation of the Notch downstream effector. RESULTS: Constitutive activation of Nrf2 signaling caused increased intestinal length along with expanded cell number and thickness of enterocytes without any alterations of secretory lineage, outcomes abrogated by concomitant disruption of Nrf2. The Nrf2 and Notch pathways in epithelium showed inverse spatial profiles, where Nrf2 activity in crypts was lower than villi. In progenitor cells of Keap1f/f::VilCre mice, Notch downstream effector Math1, which regulates a differentiation balance of cell lineage through lateral inhibition, showed suppressed expression. In vitro results demonstrated Nrf2 negatively regulated Math1, where 6 antioxidant response elements located in the regulatory regions contributed to this repression. CONCLUSIONS: Activation of Nrf2 perturbed the dialog of the Notch cascade though negative regulation of Math1 in progenitor cells, leading to enhanced enterogenesis. The crosstalk between the Nrf2 and Notch pathways could be critical for fine-tuning intestinal homeostasis and point to new approaches for the pharmacological management of absorptive deficiencies.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Intestinal Mucosa/growth & development , Intestine, Small/growth & development , NF-E2-Related Factor 2/metabolism , Regeneration/genetics , Animals , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Line , Enterocytes/drug effects , Enterocytes/physiology , Female , Intestinal Mucosa/drug effects , Intestine, Small/drug effects , Male , Mice , Models, Animal , NF-E2-Related Factor 2/agonists , NF-E2-Related Factor 2/genetics , Regeneration/drug effects , Stem Cells/drug effects , Stem Cells/physiologyABSTRACT
Intestinal failure, following extensive anatomical or functional loss of small intestine, has debilitating long-term consequences for children1. The priority of patient care is to increase the length of functional intestine, particularly the jejunum, to promote nutritional independence2. Here we construct autologous jejunal mucosal grafts using biomaterials from pediatric patients and show that patient-derived organoids can be expanded efficiently in vitro. In parallel, we generate decellularized human intestinal matrix with intact nanotopography, which forms biological scaffolds. Proteomic and Raman spectroscopy analyses reveal highly analogous biochemical profiles of human small intestine and colon scaffolds, indicating that they can be used interchangeably as platforms for intestinal engineering. Indeed, seeding of jejunal organoids onto either type of scaffold reliably reconstructs grafts that exhibit several aspects of physiological jejunal function and that survive to form luminal structures after transplantation into the kidney capsule or subcutaneous pockets of mice for up to 2 weeks. Our findings provide proof-of-concept data for engineering patient-specific jejunal grafts for children with intestinal failure, ultimately aiding in the restoration of nutritional autonomy.
Subject(s)
Intestinal Diseases/pathology , Intestinal Mucosa/transplantation , Jejunum/transplantation , Organoids/pathology , Precision Medicine/methods , Primary Cell Culture/methods , Tissue Engineering/methods , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Child , Enterocytes/pathology , Enterocytes/physiology , Enterocytes/transplantation , Extracellular Matrix/pathology , Female , HEK293 Cells , Human Umbilical Vein Endothelial Cells , Humans , Intestinal Diseases/congenital , Intestinal Diseases/therapy , Intestinal Mucosa/cytology , Intestinal Mucosa/pathology , Jejunum/cytology , Jejunum/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Proof of Concept Study , Swine , Tissue ScaffoldsABSTRACT
BACKGROUND: Recent findings by Tang et al. (2020) show dietary restriction (30%, 2 weeks) prevents methotrexate-induced mortality by modulation of the microbiota, specifically the expansion of Lactobacillus. While fundamentally insightful, upscaling this schedule is a major obstacle to clinical uptake. Here, we evaluate a safe and clinically achievable schedule of pre-therapy fasting for 48 h on microbiota composition, body composition and intestinal proliferation, and assess its impact on the severity of methotrexate-induced gastrointestinal mucositis using a validated preclinical rat model. METHODS: Age- and weight-matched male Wistar rats were treated with a sublethal dose of 45 mg/kg methotrexate with or without pre-therapy fasting. The impact of acute fasting on epithelial proliferation, body composition and the microbiota was assessed using plasma citrulline, Ki67 immunohistochemistry, miniSpec and 16S rRNA sequencing. The severity of gastrointestinal mucositis was evaluated using plasma citrulline and body weight. RESULTS: Whilst pre-therapy fasting slowed epithelial proliferation and increased microbial diversity and richness, it also induced significant weight loss and was unable to attenuate the severity of mucositis in both age- and weight-matched groups. In contrast to Tang et al., we saw no expansion of Lactobacillus following acute fasting. CONCLUSIONS: Our findings suggest that the beneficial effects of acute fasting are masked by the detrimental effects on body weight and composition and lacking influence on Lactobacillus. Future studies should consider alternative fasting schedules or aim to induce comparable microbial and mucosal manipulation without compromising body composition using clinically feasible methods of dietary or microbial intervention.
Subject(s)
Fasting , Methotrexate/toxicity , Mucositis/chemically induced , Mucositis/prevention & control , Animals , Bacteria/classification , Bacteria/growth & development , Cell Proliferation , Citrulline/blood , Enterocytes/physiology , Feces/microbiology , Gastrointestinal Microbiome , Gastrointestinal Tract/microbiology , Intestinal Mucosa/pathology , Jejunum/pathology , Lactobacillus/growth & development , Male , Rats , Rats, Wistar , Weight LossABSTRACT
Elastase-1 is one member of serine protease family, distributes in organisms widely and plays a crucial role in the invasion and development of Trichinella spiralis. In order to identify the binding of T. spiralis elastase-1 (TsEla) with host's intestinal epithelial cells (IECs) and its role in Trichinella larval intrusion, TsEla gene was cloned and expressed in our previous study. The recombinant TsEla (rTsEla) has the enzymatic activity to degrade specific peptide substrate. A specific binding between rTsEla and IECs was detected by Far Western blot and ELISA. In an in vitro invasion assay, rTsEla promoted the larval intrusion, whereas anti-rTsEla serum inhibited the larval penetration. The larval intrusion was also suppressed after the silencing of TsEla by siRNA. Silencing of TsEla gene by siRNA-291 meditated RNA interference suppressed TsEla protein expression, reduced the worm infectivity, development and reproductive capacity. These results indicated that TsEla plays an important role in the T. spiralis intrusion of host's intestinal epithelia, and it could be a prospective vaccine molecular target against T. spiralis infection.
Subject(s)
Enterocytes/physiology , Helminth Proteins/metabolism , Intestinal Mucosa/physiology , Pancreatic Elastase/metabolism , Trichinella spiralis/enzymology , Trichinellosis/parasitology , Animals , Enterocytes/immunology , Epithelial Cells , Gene Expression Regulation, Enzymologic , Helminth Proteins/immunology , Intestines , Larva/physiology , Mice , Mice, Inbred BALB C , Pancreatic Elastase/chemistry , Pancreatic Elastase/genetics , Prospective Studies , Trichinella spiralis/genetics , Trichinellosis/immunologyABSTRACT
In hematophagous insects, the midgut is a fundamental barrier against infections and limits the development and transmission of pathogens. However, in mosquitoes, cell differentiation, proliferation, and cell cycle process in the midgut have not been characterized. Here we provide evidence of how cell cycle progression occurs in the newly emerged Anopheles albimanus mosquito midgut and describing cyclins expression as mediators of the cell cycle. The cell cycle at different post-emergence times was evaluated in disaggregated cells from midgut tissue using flow cytometry. Also, cyclins A, B, and E were identified by bioinformatics tools. These cyclins were used to analyze cell cycle progression. Flow cytometry data and the expression-pattern of the cyclins by qRT-PCR supported a polyploidy process, besides mitosis marker was marginally detected and only in newly emerged mosquitoes. Our results suggest that DNA increment in midguts occurs by polyploidy during the first hours post-emergence.
Subject(s)
Anopheles/physiology , Cyclins/metabolism , Enterocytes/physiology , Insect Proteins/metabolism , Intestines/cytology , Animals , Cell Cycle , Cells, Cultured , Computational Biology , Cyclins/genetics , DNA Replication , Flow Cytometry , Gene Expression Regulation, Developmental , Insect Proteins/genetics , Life Cycle Stages , Phylogeny , PolyploidyABSTRACT
A novel coronavirus-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-emerged in humans in Wuhan, China, in December 2019 and has since disseminated globally1,2. As of April 16, 2020, the confirmed case count of coronavirus disease 2019 (COVID-19) had surpassed 2 million. Based on full-genome sequence analysis, SARS-CoV-2 shows high homology to SARS-related coronaviruses identified in horseshoe bats1,2. Here we show the establishment and characterization of expandable intestinal organoids derived from horseshoe bats of the Rhinolophus sinicus species that can recapitulate bat intestinal epithelium. These bat enteroids are fully susceptible to SARS-CoV-2 infection and sustain robust viral replication. Development of gastrointestinal symptoms in some patients with COVID-19 and detection of viral RNA in fecal specimens suggest that SARS-CoV-2 might cause enteric, in addition to respiratory, infection3,4. Here we demonstrate active replication of SARS-CoV-2 in human intestinal organoids and isolation of infectious virus from the stool specimen of a patient with diarrheal COVID-19. Collectively, we established the first expandable organoid culture system of bat intestinal epithelium and present evidence that SARS-CoV-2 can infect bat intestinal cells. The robust SARS-CoV-2 replication in human intestinal organoids suggests that the human intestinal tract might be a transmission route of SARS-CoV-2.
Subject(s)
Betacoronavirus/pathogenicity , Coronavirus Infections/pathology , Coronavirus Infections/transmission , Intestines/virology , Organoids/virology , Pneumonia, Viral/pathology , Pneumonia, Viral/transmission , Animals , COVID-19 , Cell Differentiation , Cells, Cultured , Child, Preschool , Chiroptera/virology , Chlorocebus aethiops , Coronavirus Infections/virology , Enterocytes/pathology , Enterocytes/physiology , Enterocytes/virology , Female , Humans , Infant , Intestinal Mucosa/pathology , Intestinal Mucosa/virology , Intestines/pathology , Male , Organoids/pathology , Pandemics , Pneumonia, Viral/virology , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2 , Vero Cells , Viral Load/genetics , Viral Load/methods , Viral Tropism/physiologySubject(s)
Enterocytes/physiology , Intestinal Mucosa/physiology , Multipotent Stem Cells/physiology , Protein Kinase C/metabolism , Animals , Cell Proliferation/drug effects , Cells, Cultured , Enterocytes/drug effects , Intestinal Mucosa/cytology , Mice , Mice, Transgenic , Multipotent Stem Cells/drug effects , Organoids , Primary Cell Culture , Protein Kinase C/genetics , Pyrimidines , Signal Transduction/drug effectsABSTRACT
BACKGROUND AND AIMS: Intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. Disruption of this homeostasis is associated with disorders such as inflammatory bowel disease (IBD). We investigated the role of Sirtuin 2 (SIRT2), a NAD-dependent protein deacetylase, in intestinal epithelial cell (IEC) proliferation and differentiation and the mechanism by which SIRT2 contributes to maintenance of intestinal cell homeostasis. METHODS: IECs were collected from SIRT2-deficient mice and patients with IBD. Expression of SIRT2, differentiation markers (mucin2, intestinal alkaline phosphatase, villin, Na,K-ATPase, and lysozyme) and Wnt target genes (EPHB2, AXIN2, and cyclin D1) was determined by western blot, real-time RT-PCR, or immunohistochemical (IHC) staining. IECs were treated with TNF or transfected with siRNA targeting SIRT2. Proliferation was determined by villus height and crypt depth, and Ki67 and cyclin D1 IHC staining. For studies using organoids, intestinal crypts were isolated. RESULTS: Increased SIRT2 expression was localized to the more differentiated region of the intestine. In contrast, SIRT2 deficiency impaired proliferation and differentiation and altered stemness in the small intestinal epithelium ex vivo and in vivo. SIRT2-deficient mice showed decreased intestinal enterocyte and goblet cell differentiation but increased the Paneth cell lineage and increased proliferation of IECs. Moreover, we found that SIRT2 inhibits Wnt/ß-catenin signaling, which critically regulates IEC proliferation and differentiation. Consistent with a distinct role for SIRT2 in maintenance of gut homeostasis, intestinal mucosa from IBD patients exhibited decreased SIRT2 expression. CONCLUSION: We demonstrate that SIRT2, which is decreased in intestinal tissues from IBD patients, regulates Wnt-ß-catenin signaling and is important for maintenance of IEC proliferation and differentiation.
Subject(s)
Colitis, Ulcerative/pathology , Crohn Disease/pathology , Enterocytes/physiology , Goblet Cells/physiology , Sirtuin 2/metabolism , Animals , Biopsy , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Colon/cytology , Colon/pathology , Colonoscopy , Humans , Mice , Mice, Knockout , Organoids , Primary Cell Culture , Sirtuin 2/analysis , Sirtuin 2/genetics , Wnt Signaling PathwayABSTRACT
Metabolites are increasingly appreciated for their roles as signaling molecules. To dissect the roles of metabolites, it is essential to understand their signaling pathways and their enzymatic regulations. From an RNA interference (RNAi) screen for regulators of intestinal stem cell (ISC) activity in the Drosophila midgut, we identified adenosine receptor (AdoR) as a top candidate gene required for ISC proliferation. We demonstrate that Ras/MAPK and Protein Kinase A (PKA) signaling act downstream of AdoR and that Ras/MAPK mediates the major effect of AdoR on ISC proliferation. Extracellular adenosine, the ligand for AdoR, is a small metabolite that can be released by various cell types and degraded in the extracellular space by secreted adenosine deaminase. Interestingly, down-regulation of adenosine deaminase-related growth factor A (Adgf-A) from enterocytes is necessary for extracellular adenosine to activate AdoR and induce ISC overproliferation. As Adgf-A expression and its enzymatic activity decrease following tissue damage, our study provides important insights into how the enzymatic regulation of extracellular adenosine levels under tissue-damage conditions facilitates ISC proliferation.
Subject(s)
Adenosine Deaminase/metabolism , Drosophila Proteins/metabolism , Enterocytes/physiology , Multipotent Stem Cells/physiology , Receptors, Purinergic P1/metabolism , Adenosine/metabolism , Animals , Animals, Genetically Modified , Cell Differentiation , Cell Proliferation , Down-Regulation , Drosophila , Drosophila Proteins/genetics , Gene Knock-In Techniques , Gene Knockdown Techniques , MAP Kinase Signaling System/genetics , RNA Interference , Receptors, Purinergic P1/geneticsABSTRACT
Small intestinal epithelium homeostasis involves four principal cell types: enterocytes, goblet, enteroendocrine and Paneth cells. Epidermal growth factor (EGF) has been shown to affect enterocyte differentiation. This study determined the effect of dietary EGF on goblet, enteroendocrine and Paneth cell differentiation in piglet small intestine and potential mechanisms. Forty-two weaned piglets were used in a 2 × 3 factorial design; the major factors were time post-weaning (days 7 and 14) and dietary treatment (0, 200 or 400 µg/kg EGF supplementation). The numbers of goblet and enteroendocrine cells were generally greater with the increase in time post-weaning. Moreover, the supplementation of 200 µg/kg EGF increased (P < 0.01) the number of goblet and enteroendocrine cells in villus and crypt of the piglet small intestine as compared with the control. Dietary supplementation with 200 µg/kg EGF enhanced (P < 0.05) abundances of differentiation-related genes atonal homologue 1, mucin 2 and intestinal trefoil factor 3 messenger RNA (mRNA) as compared with the control. Piglets fed 200 or 400 µg/kg EGF diet had increased (P < 0.05) abundances of growth factor-independent 1, SAM pointed domain containing ETS transcription factor and pancreatic and duodenal homeobox 1 mRNA, but decreased the abundance (P < 0.01) of E74 like ETS transcription factor 3 mRNA as compared with the control. Animals receiving 400 µg/kg EGF diets had enhanced (P < 0.05) abundances of neurogenin3 and SRY-box containing gene 9 mRNA as compared with the control. The mRNA abundance and protein expression of lysozyme, a marker of Paneth cell, were also increased (P < 0.05) in those animals. As compared with the control, dietary supplementation with 200 µg/kg EGF increased the abundance of EGF receptor mRNA and the ratio of non-phospho(p)-ß-catenin/ß-catenin (P < 0.05) in villus epithelial cells at days 7 and 14. This ratio in crypt epithelial cells was higher (P < 0.05) on the both 200 and 400 µg/kg EGF groups during the same period. Our results demonstrated that dietary EGF stimulated goblet, enteroendocrine and Paneth cell differentiation in piglets during the post-weaning period, partly through EGFR and Wnt/ß-catenin signalling.
Subject(s)
Dietary Supplements/analysis , Epidermal Growth Factor/administration & dosage , Swine/physiology , Wnt Proteins/metabolism , Wnt Signaling Pathway , beta Catenin/metabolism , Animals , Cell Differentiation , Diet/veterinary , Enterocytes/physiology , Epithelial Cells/physiology , ErbB Receptors/genetics , ErbB Receptors/metabolism , Intestinal Mucosa/physiology , Intestine, Small/physiology , RNA, Messenger/genetics , Swine/genetics , Weaning , Wnt Proteins/genetics , beta Catenin/geneticsABSTRACT
The aim of the present study was to elucidate the in vitro short-term (2-h) and longer-term (24-h) effects of hyperosmolar media (500 and 680 mOsm/L) on intestinal epithelial cells using the human colonocyte Caco-2 cell line model. We found that a hyperosmolar environment slowed down cell proliferation compared to normal osmolarity (336 mOsm/L) without inducing cell detachment or necrosis. This was associated with a transient reduction of cell mitochondrial oxygen consumption, increase in proton leak, and decrease in intracellular ATP content. The barrier function of Caco-2 monolayers was also transiently affected since increased paracellular apical-to-basal permeability and modified electrolyte permeability were measured, allowing partial equilibration of the trans-epithelial osmotic difference. In addition, hyperosmotic stress induced secretion of the pro-inflammatory cytokine IL-8. By measuring expression of genes involved in energy metabolism, tight junction forming, electrolyte permeability and intracellular signaling, different response patterns to hyperosmotic stress occurred depending on its intensity and duration. These data highlight the potential impact of increased luminal osmolarity on the intestinal epithelium renewal and barrier function and point out some cellular adaptive capacities towards luminal hyperosmolar environment.
Subject(s)
Cell Proliferation , Enterocytes/metabolism , Mitochondria/metabolism , Osmolar Concentration , Oxygen Consumption , Caco-2 Cells , Enterocytes/physiology , Humans , Interleukin-8/metabolism , Intestinal Mucosa/metabolism , Intestinal Mucosa/physiopathology , Signal TransductionABSTRACT
The intestinal epithelium undergoes constant regeneration driven by intestinal stem cells. How old age affects the transcriptome in this highly dynamic tissue is an important, but poorly explored question. Using transcriptomics on sorted intestinal stem cells and adult enterocytes, we identified candidate genes, which change expression on aging. Further validation of these on intestinal epithelium of multiple middle-aged versus old-aged mice highlighted the consistent up-regulation of the expression of the gene encoding chemokine receptor Ccr2, a mediator of inflammation and several disease processes. We observed also increased expression of Strc, coding for stereocilin, and dramatically decreased expression of Rps4l, coding for a ribosome subunit. Ccr2 and Rps4l are located close to the telomeric regions of chromosome 9 and 6, respectively. As only few genes were differentially expressed and we did not observe significant protein level changes of identified ageing markers, our analysis highlights the overall robustness of murine intestinal epithelium gene expression to old age.