Intestinal Epithelial Function Research Articles

Epithelial Skiv2l deletion promotes aberrant secretory cell differentiation in the small intestine

Background: Inflammatory bowel disease (IBD) remains a global concern, with millions affected and incidence increasing. The etiology of IBD is multifaceted, but gene variants in epithelial and immune cells can contribute to its onset. Trichohepatoenteric syndrome (THES) is a form of monogenic very early onset IBD (VEO-IBD) that arises due to variants in SKIV2L and TTC37, which are components of the cytoplasmic RNA exosome involved in mRNA quality control. Patients with THES experience a decreased quality of life characterized by poor growth, intractable diarrhea, and immune defects. There are limited drugs approved by the FDA for treatment of IBD in children and those that have been approved are geared towards suppressing the immune system and have side effects. Therefore, there is a need for the development of therapeutics that target epithelial regeneration and repair. Understanding how genetic variants, such as those observed in THES, are involved in epithelial function is one avenue to improved therapies. The objective of the current study is to understand how the RNA exosome contributes to intestinal epithelial homeostasis by studying deletion of SKIV2L. Preclinical studies in mice revealed that global Skiv2l deletion resulted in increased mTOR signaling, and overactivated T cells, while B cell specific deletion of Skiv2l led to impaired B cell development. Ex vivo studies from our lab revealed that colonoids from THES patients exhibit decreased organoid formation effciency (OFE), suggesting that SKIV2L may have important roles in the intestinal stem cell self-renewal. We hypothesize that SKIV2L is required for intestinal epithelial cell growth and tissue function. Methods: We generated VillinCre;Skiv2l ( Skiv2l KO) mice with Skiv2l deletion in intestinal epithelial cells. Skiv2l WT and KO mice weights were monitored weekly, starting from ~2 weeks of age. Between 4 and 6 weeks of age, intestines were harvested for immunofluorescence, flow cytometry and OFE assays. Results and conclusion: Skiv2l KO mice experienced failure to thrive, were significantly smaller, and had shorter colons and small intestines compared to their WT counterparts. Immunofluorescence and flow cytometry revealed increased secretory cells in the small intestine of Skiv2l KO compared to WT mice, with significant accumulation of goblet cells in the crypt region in KO mice. Furthermore, Skiv2l KO mice had reduced OFE. These findings suggest that SKIV2L is essential for intestinal homeostasis and that aberrant secretory cell differentiation may underlie the decreased stem cell self-renewal observed in Skiv2l KO mice and THES patients. More broadly, these studies suggest an essential role for mRNA quality control in normal intestinal homeostasis. University of Pennsylvania, Institution of Regenerative Medicine; NIH R01DK124369. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Modeling the cell biology of monogenetic intestinal epithelial disorders.

Monogenetic variants are responsible for a range of congenital human diseases. Variants in genes that are important for intestinal epithelial function cause a group of disorders characterized by severe diarrhea and loss of nutrient absorption called congenital diarrheas and enteropathies (CODEs). CODE-causing genes include nutrient transporters, enzymes, structural proteins, and vesicular trafficking proteins in intestinal epithelial cells. Several severe CODE disorders result from the loss-of-function in key regulators of polarized endocytic trafficking such as the motor protein, Myosin VB (MYO5B), as well as STX3, STXBP2, and UNC45A. Investigations of the cell biology and pathophysiology following loss-of-function in these genes have led to an increased understanding of both homeostatic and pathological vesicular trafficking in intestinal epithelial cells. Modeling different CODEs through investigation of changes in patient tissues, coupled with the development of animal models and patient-derived enteroids, has provided critical insights into the enterocyte differentiation and function. Linking basic knowledge of cell biology with the phenotype of specific patient variants is a key step in developing effective treatments for rare monogenetic diseases. This knowledge can also be applied more broadly to our understanding of common epithelial disorders.

Establishing porcine jejunum-derived intestinal organoids to study the function of intestinal epithelium as an alternative for animal testing

Establishing porcine jejunum-derived intestinal organoids to study the function of intestinal epithelium as an alternative for animal testing

Muramyl dipeptide causes mitochondrial dysfunction and intestinal inflammatory cytokine responses in rats.

Intestinal flora and the translocation of its products, such as muramyl dipeptide (MDP), are common causes of sepsis. MDP is a common activator of the intracellular pattern recognition receptor NOD2, and MDP translocation can cause inflammatory damage to the small intestine and systemic inflammatory responses in rats. Therefore, this study investigated the effects of MDP on the intestinal mucosa and distant organs during sepsis and the role of the NOD2/AMPK/LC3 pathway in MDP-induced mitochondrial dysfunction in the intestinal epithelium. Fifty male Sprague Dawley rats were randomly divided into five treatment groups: lipopolysaccharide (LPS) only, 1.5 and 15 mg/kg MDP + LPS, and 1.5 and 15 mg/kg MDP + short-peptide enteral nutrition (SPEN) + LPS. The total caloric intake was the same per group. The rats were euthanized 24 hours after establishing the model, and peripheral blood and small intestinal mucosal and lung tissues were collected. Compared to the LPS group, both MDP + LPS groups had aggravated inflammatory damage to the intestinal mucosal and lung tissues, increased IL-6 and MDP production, increased NOD2 expression, decreased AMPK and LC3 expression, increased mitochondrial reactive oxygen species production, and decreased mitochondrial membrane potential. Compared to the MDP + LPS groups, the MDP + SPEN+LPS groups had decreased IL-6 and MDP production, increased AMPK and LC3 protein expression, and protected mitochondrial and organ functions. MDP translocation reduced mitochondrial autophagy by regulating the NOD2/AMPK/LC3 pathway, causing mitochondrial dysfunction. SPEN protected against MDP-induced impairment of intestinal epithelial mitochondrial function during sepsis.

Effect of dietary l-glutamine supplementation on the intestinal physiology and growth during Solea senegalensis larval development

The maturation of the intestinal digestive and absorptive functions might limit the amount of absorbed nutrients to fulfil the high requirements of the fast-growing marine fish larva. Glutamine (Gln) has been described to improve intestinal epithelium functions, due to its involvement in energy metabolism and protein synthesis. The purpose of this study was to evaluate dietary 0.2% Gln supplementation on aspects of intestinal physiology, protein metabolism and growth-related genes expression in Senegalese sole larvae. Experiment was carried out between 12 and 33 days post hatching (DPH) and fish were divided into two experimental groups, one fed Artemia spp. (CTRL) and the other fed Artemia spp. supplemented with Gln (GLN). GLN diet had two times more Gln than the CTRL diet. Samples were collected at 15, 19, 26 and 33 DPH for biometry, histology, and digestive enzymes activity, and at 33 DPH for gene expression, protein metabolism and AA content determination. Growth was significantly higher for Senegalese sole fed GLN diet, supported by differences on protein metabolism and growth-related gene expression. Slight differences were observed between treatments regarding the intestinal physiology. Overall, GLN diet seems to be directed to enhance protein metabolism leading to higher larval growth.

Neuregulin 1 (NRG1) and its receptors in the enteric nervous system and other parts of the gastrointestinal wall.

Neuregulin 1 (NRG1) belonging to the transmembrane growth factors family is widespread in living organisms. It acts through ErbB family receptors and first of all takes part in embryogenesis, as well as in developmental, regenerative and adaptive processes occurring in various internal organs and systems. It is known that NRG1 and its receptors are present in various parts of the gastrointestinal (GI) tract. First of all NRG1 and ErbB receptors have been detected in the enteric nervous system (ENS) localized in the wall of the esophagus, stomach and intestine and regulating the majority of the GI tract functions, but also in the mucosal and muscular layers of the GI tract. The NRG1/ErbB pathway is involved in the development and differentiation of the ENS and regulation of the intestinal epithelium functions. Moreover, dysregulation of this pathway results in a wide range of gastrointestinal diseases. However, till now there are no summarizations of previous studies concerning distribution and functions of NRG1 and its receptors in the GI tract. The present review fills this gap.

In-Ovo Glutamine Administration Enhances Intestinal Development and Functions in Broiler Chickens: Insights from Enteroid Models

BackgroundEarly life events play significant roles in tissue development and animal health in their later life. Early nutrition, through in-ovo delivery, has shown beneficial effects on improving intestinal health in broiler chickens. However, the underlying mechanism is not fully investigated. A recently developed enteroid culture technique allows investigations on intestinal epithelial functions that are close to physiologic conditions. ObjectivesIn this study, we evaluated the short- and long-term effects of in-ovo administration of glutamine (Gln) on intestinal epithelial development and functions by using intestinal enteroid culture and tissue electrophysiologic analysis. MethodsA hundred eggs of commercial Cobb500 broilers were in-ovo injected with 0.2 mL of either phosphate-buffered saline (PBS) or 3% Gln at embryonic day 18 (E18). Chicks were killed on the day of hatch, and at 3- and 14-d posthatch. Enteroids were generated from the small intestine. After 4 d of culture, enteroids were harvested for 5-ethynyl-2′-deoxyuridine proliferation, fluorescein isothiocyanate-4 kDa dextran permeability, and glucose absorption assays. At day 3 (d3) and day 14 (d14), intestinal barrier and nutrient transport functions were measured by the Ussing chamber. The gene expression of epithelial cell markers, nutrient transporters, and tight-junction proteins were analyzed in both intestinal tissues and enteroids. ResultsIn comparison with the PBS control group, in-ovo Gln increased intestinal villus morphology, epithelial cell proliferation, and differentiation, and altered epithelial cell population toward increased number of enteroendocrine and goblet cells while decreasing Paneth cells. Enteroids gene expression of nutrient transporters (B0AT1, SGLT1, and EAAT3), tight junction (ZO2), glucose absorption, and barrier functions were enhanced on the day of hatch. Long-term increases of intestinal di-peptide and alanine transport were observed at day 14 posthatch. ConclusionsTogether our results suggested that the in-ovo injection of Gln stimulated intestinal epithelium proliferation and programmed the epithelial cell differentiation toward absorptive cells.

Human intestinal organoids: Modeling gastrointestinal physiology and immunopathology - current applications and limitations.

Human intestinal organoids are an ideal model system for studying gastrointestinal physiology and immunopathology. Altered physiology and mucosal immune response are hallmarks of numerous intestinal functional and inflammatory diseases, including inflammatory bowel disease (IBD), coeliac disease, irritable bowel syndrome (IBS), and obesity. These conditions impact the normal epithelial functions of the intestine, such as absorption, barrier function, secretion, and host-microbiome communication. They are accompanied by characteristic intestinal symptoms and have significant societal, economic, and healthcare burdens. To develop new treatment options, cutting-edge research is required to investigate their etiology and pathology. Human intestinal organoids derived from patient tissue recapitulate the key physiological and immunopathological aspects of these conditions, providing a promising platform for elucidating disease mechanisms. This review will summarize recent reports on patient-derived human small intestinal and colonic organoids and highlight how these models have been used to study intestinal epithelial functions in the context of inflammation, altered physiology, and immune response. Furthermore, it will elaborate on the various organoid systems in use and the techniques/assays currently available to study epithelial functions. Finally, it will conclude by discussing the limitations and future perspectives of organoid technology.

Reduced LHFPL3-AS2 lncRNA expression is linked to altered epithelial polarity and proliferation, and to ileal ulceration in Crohn disease

Disruption of intestinal epithelial functions is linked to Crohn disease (CD) pathogenesis. We identified a widespread reduction in the expression of long non-coding RNAs (lncRNAs) including LHFPL3-AS2 in the treatment-naïve CD ileum of the RISK pediatric cohort. We validated the reduction of LHFPL3-AS2 in adult CD and noted a further reduction in patients with more severe CD from the RISK cohort. LHFPL3-AS2 knockdown in Caco-2 cells robustly affected epithelial monolayer morphogenesis with markedly reduced confluency and spreading, showing atypical rounding, and clumping. mRNA-seq analysis of LHFPL3-AS2 knockdown cells highlighted the reduction of genes and pathways linked with apical polarity, actin bundles, morphogenesis, and the b-catenin-TCF4 complex. LHFPL3-AS2 knockdown significantly reduced the ability of cells to form an internal lumen within the 3-dimensional (3D) cyst model, with mislocalization of actin and adherent and tight junction proteins, affecting epithelial polarity. LHFPL3-AS2 knockdown also resulted in defective mitotic spindle formation and consequent reduction in epithelial proliferation. Altogether, we show that LHFPL3-AS2 reduction affects epithelial morphogenesis, polarity, mitotic spindle formation, and proliferation, which are key processes in maintaining epithelial homeostasis in CD. Reduced expression of LHFPL3-AS2 in CD patients and its further reduction with ileal ulceration outcome, emphasizes its significance in this context.

PSV-8 Early Life Adversity Impacts Intestinal Epithelial Development and Stem Cells Activities in Pigs

Abstract Early life adversity significantly impacts organ development and predisposes animals towards greater risk of diseases later in life. In the previous research in pigs, long lasting intestinal barrier dysfunctions have been found to be associated with early weaning stress. However, the underlying mechanism is not fully elucidated. Intestinal epithelial functions are associated with the number and maturation of multicellular components, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells (EEC). Intestinal epithelial stem cells (IESC) give rise to the whole epithelium within 5 to 7 days. Such rapid turnover rate allows intestinal adaptation to stress conditions. We hypothesized that the early life stress impacted stem cells activities which would contribute to the long-lasting post-stress functional changes in intestinal epithelium. The objective of the present study is to evaluate the epithelium component cells and IESC proliferation and differentiation activities in an early weaning stress model in pigs. Piglets (n = 18) were obtained from commercial litters and were pair-weaned at the age of d 15 (early weaning, EW) and d 26 (late weaning, LW). Piglets were co-housed and raised under the same conditions. At the age of d 35, ileal tissues were isolated and fixed for morphological measurements. Intestinal crypts were isolated and cultured in the Matrigel matrix to form enteroids. The proliferation activities of IESCs were measured by enteroids morphology; differentiation capacity was measured by quantification of epithelial cell markers with and without chemicals-induced enteroids differentiation. Gene expressions and (or) staining of specific markers for IESCs (Olfm4, BMI1), enterocytes (ALP1), goblet cells (Muc2), Paneth cells (LYZ), and EECs (ChgA) in ileal tissue and ileal enteroids were also quantified. Data were analyzed by the student’s t-test. Our results showed that EW significantly (P < 0.05) reduced ileal villus width and crypt fission rate, while no changes were found on villus height and crypt depth compared with LW control. The alterations of ileal epithelium components in EW pigs were indicated by significantly fewer number of EECs, greater number of the goblet cells, and reduced ALP1 marker expression. The enteroids formation and budding rates were both significantly suppressed in EW pigs, which indicated a reduced IESC proliferating activity. In the fully developed enteroids, gene expressions of LYZ, ALP1, BMI1, Olfm4, and Hes1 were least in EW, but Muc2 expression was greater compared with control. In the differentiation culture media, the enteroids from EW pigs exhibited significantly enhanced potential for goblet cell differentiation but reduced capacity for enterocyte differentiation compared with LW pigs. Overall, the results indicate that early weaning stress affects the programming and activity of IESCs, which leads to an altered epithelium cell population and intestinal functions. Further understanding of mechanisms regulating IESCs activity will shed light on novel strategies mitigating stress-associated intestinal dysfunctions.

Modulation of pectin on intestinal barrier function via changes in microbial functional potential and bile acid metabolism

Weaning is one of the major factors that cause stress and intestinal infection in infants and in young animals due to an immature intestine and not fully developed immune functions. Pectin (PEC), a prebiotic polysaccharide, has attracted considerable attention in intestinal epithelial signaling and function via modulation of the microbial community. A total of 16 weaned piglets (21-d-old) were randomly assigned into two groups: control group and PEC group. Supplementation of 5% pectin improved intestinal mucosal barrier function by modulating the composition of the bile acid pool in piglets. Specifically, piglets in PEC group had less serum D-lactate content and alkaline phosphatase activity. In the ileum, dietary pectin increased the number of crypt PAS/AB-positive goblet cells and the mRNA expressions of MUC2, ZO-1, and Occludin. Piglets in PEC group displayed a decreased abundance of Enterococcus (2.71 vs. 65.92%), but the abundances of Lactobacillus (30.80 vs. 7.93%), Streptococcus (21.41 vs. 14.81%), and Clostridium_sensu_stricto_1 (28.34 vs. 0.01%) were increased. Elevated concentrations of bile acids especially hyocholic acid species (HCAs) including HCA, HDCA, and THDCA were also observed. Besides, correlation analysis revealed that dietary pectin supplementation may have beneficial effects through stimulation of the crosstalk between gut microbes and bile acid synthesis within the enterohepatic circulation. Thus, dietary pectin supplementation exhibited a further positive effect on the healthy growth and development of weaned piglets. These findings suggest pectin supplementation as the prebiotic is beneficial for gut health and improvement of weaned stress via regulating microbiota and bile acid metabolism.

Curculigoside mitigates dextran sulfate sodium‑induced colitis by activation of KEAP1‑NRF2 interaction to inhibit oxidative damage and autophagy of intestinal epithelium barrier.

Curculigoside (CUR), a primary active ingredient of Curculigo orchioides Gaertn, serves an important role in the intervention of numerous diseases, including ulcerative colitis, rheumatoid arthritis, myocardial ischemia, etc. However its specific mechanisms of therapy have not been fully elucidated. The aim of the present study was to elucidate the mechanisms underlying the anti‑oxidative stress and anti‑ulcerative colitis (UC) effects of CUR. Mouse model of dextran sulfate sodium (DSS)‑induced colitis, along with Caco2 and mouse intestine organoid invitro models were used. The effect of CUR on mitigating the symptoms of chronic colitis was investigated. Through ELISA experiments, it was observed that CUR alleviated the inflammation status in mice with chronic colitis. This was evidenced by the downregulation of inflammatory cytokines such as TNF‑α and IL‑6 and ‑1β and decreased neutrophil infiltration along with downregulated myeloperoxidase activity. CUR helped in maintaining the barrier functions of intestinal epithelium. Invitro TNF‑α stimulation of organoids and H2O2 stimulation of Caco2 cells demonstrated the capabilities of CUR to rescue cells from oxidative stress. There was activation of Nrf2 both invivo and invitro, accompanied by enhanced autophagy. Mechanistic studies of cells and Nrf2 knockout mice demonstrated that Nrf2 served a pivotal role in inhibition of UC by curculigoside via interaction with Kelch‑like ECH‑associated protein 1 (Keap1). Invitro and invivo experiments confirmed that CUR activated Nrf2 via Keap1/Nrf2 interaction, resulting in decreased oxidative stress and promoted autophagy. These findings demonstrated that CUR could effectively mitigate colitis and may have clinical application in UC therapy.

Effect of Different Dietary Lipid Sources on Growth Performance, Nutrient Digestibility, and Intestinal Health in Weaned Pigs.

To investigate the effects of lipid sources on growth performance and intestinal health, 72 weaned pigs were randomly allocated to three treatments. Pigs were fed with a corn-soybean meal diet containing 2% soybean oil (SO), or fish-palm-rice oil mixture (FPRO), or coconut-palm-rice oil mixture (CPRO). The trial lasted for 28 days; blood and intestinal tissue samples were collected. The results showed that the crude fat digestibility of the FPRO group was higher than that of the SO and CPRO groups (p < 0.05). The FPRO group also had higher digestibility of dry matter, ash, and gross energy than the SO group (p < 0.05); compared to the SO group, the serum interlukin-6 (IL-6) concentration was decreased. Interestingly, the FPRO and CPRO groups had higher villus height than the SO group in the jejunum and ileum, respectively (p < 0.05). Moreover, the FPRO group had higher Lactobacillus abundance than the SO group in the colon and cecum (p < 0.05). Importantly, the expression levels of tight junction protein ZO-1, Claudin-1, and Occludin in the duodenal and ileal mucosa were higher in the FPRO group than in the SO and CPRO groups (p < 0.05). The expression levels of nutrient transporters such as the CAT-1, PepT1, FATP1, and SGLT1 were higher in the FPRO group than in the SO group (p < 0.05). The improved digestibility and intestinal epithelium functions, as well as the reduced inflammatory cytokines, in the FPRO and CPRO group suggest that a mixed lipid source such as the FPRO deserves further attention.

Arula-7 powder improves diarrhea and intestinal epithelial tight junction function associated with its regulation of intestinal flora in calves infected with pathogenic Escherichia coli O1

BackgroundThe effects of Arula-7 powder (ASP) on diarrhea and intestinal barrier function associated with its regulation of intestinal microflora in calves infected with pathogenic Escherichia coli O1 (E. coli O1) were studied.MethodTwenty Holstein calves were randomly divided into four treatment groups: normal control (NC), model control (MC), 0.5 mg/kg ciprofloxacin (CIP) and 2.50 g/kg ASP groups.ResultsASP inhibited the relative abundance of Proteobacteria, Selenomonadales, and Enterobacteriales, and increased the relative abundance of Lactobacillus, Faecalibacterium, and Alloprevotella. Moreover, we demonstrated for the first time that the ASP and CIP promoted weight gain, reduced the diarrhea rate (P < 0.05), and enhanced antioxidant capacity (P < 0.05) due to the increase in average daily gain (ADG), total protein (TP), and albumin (ALB). In addition, ASP and CIP increased the expression of Zunola occludens-1 (ZO-1), Occludin, and Claudin-1 in the ileum (P < 0.05), and improved immunity due to increase levels of interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-γ (IFN-γ), immunoglobulin A (IgA), and immunoglobulin G (IgG) in the serum, strengthened CD4+T levels in the ileal mucosa and reducing CD8+T and CD11c+T (P < 0.05).ConclusionHence, The intestinal microbiota environment formed by early intervention of ASP powder has a protective effect on the intestinal mucosal function of calves infected with pathogenic E. coli.4nw2EB1YynqDa4Tsj5-kjPVideo

Influences of wheat bran fiber on growth performance, nutrient digestibility, and intestinal epithelium functions in Xiangcun pigs

Dietary fiber (DF) has long been looked as an essential “nutrients” both for animals and humans as it can promote the intestinal tract development and modulate the intestinal epithelium functions and the gut microbiota. This study was conducted to investigate the influences of wheat bran fiber (WBF) on growth performance and intestinal epithelium functions in Xiangcun pigs. Twenty Xiangcun pigs with 60 days of age were divided to two groups and exposed to a basal diet (BD) or BD containing 4.3% wheat bran fiber (WFD). WFD improved the average daily gain (ADG) and feed-to-gain ratio (F:G) (p < 0.01). Moreover, WFD lowered the serum triglyceride (TC), d-lactate, and malonicdialdehyde (MDA) concentrations, but significantly improved the glutathione (GSH) activity and total antioxidant capacity (T-AOC) (p < 0.05). Interestingly, WFD observably improved the villus height (VH) and the villus height to crypt depth ratio (V/C) in the small intestine (p < 0.05). The jejunal sucrase and ileal maltase activities were higher in the WFD group (p < 0.05). WFD markedly elevated the tight junction protein ZO-1 and claudin-1 expression levels in the jejunum and ileum (p < 0.05). The sodium/glucose co-transporter 1 (SGLT1), glucose transporter 2 (GLUT2), and fatty acid transport proteins 4 (FATP-4) expression levels in jejunum and ileum were also elevated under WFD (p < 0.05). WFD decreased the IL-6 impression level in the duodenum and ileum, but significantly increased the IL-10 expression levels in jejunum and ileum (p < 0.05). Moreover, WFD reduced the abundance of E. coli, but elevated the abundances of beneficial microorganisms (e.g. Lactobacillus and Bacillus) and the production microbial metabolites (e.g. propionic acid and butyrate acid) in the cecum (p < 0.05).

GATA6-AS1 Regulates Intestinal Epithelial Mitochondrial Functions, and its Reduced Expression is Linked to Intestinal Inflammation and Less Favourable Disease Course in Ulcerative Colitis.

Widespread dysregulation of long non-coding RNAs [lncRNAs] including a reduction in GATA6-AS1 was noted in inflammatory bowel disease [IBD]. We previously reported a prominent inhibition of epithelial mitochondrial functions in ulcerative colitis [UC]. However, the connection between reduction of GATA6-AS1 expression and attenuated epithelial mitochondrial functions was not defined. Mucosal transcriptomics was used to conform GATA6-AS1 reduction in several treatment-naïve independent human cohorts [n=673]. RNA pull-down followed by mass spectrometry was used to determine the GATA6-AS1 interactome. Metabolomics and mitochondrial respiration following GATA6-AS1 silencing in Caco-2 cells were used to elaborate on GATA6-AS1 functions. GATA6-AS1 showed predominant expression in gut epithelia using single cell datasets. GATA6-AS1 levels were reduced in Crohn's disease [CD] ileum and UC rectum in independent cohorts. Reduced GATA6-AS1 lncRNA was further linked to a more severe UC form, and to a less favourable UC course. The GATA6-AS1 interactome showed robust enrichment for mitochondrial proteins, and included TGM2, an autoantigen in coeliac disease that is induced in UC, CD and coeliac disease, in contrast to GATA6-AS1 reduction in these cohorts. GATA6-AS1 silencing resulted in induction of TGM2, and this was coupled with a reduction in mitochondrial membrane potential and mitochondrial respiration, as well as in a reduction of metabolites linked to aerobic respiration relevant to mucosal inflammation. TGM2 knockdown in GATA6-AS1-deficient cells rescued mitochondrial respiration. GATA6-AS1 levels are reduced in UC, CD and coeliac disease, and in more severe UC forms. We highlight GATA6-AS1 as a target regulating epithelial mitochondrial functions, potentially through controlling TGM2 levels.

Mechanisms of epithelial growth and development in the zebrafish intestine.

The intestinal epithelium is a complex tissue monolayer composed of regionally and functionally specialized intestinal epithelial cells. Given epithelial exposure to harsh and varied luminal conditions, epithelial cells continuously regenerate to sustain the barrier against environmental factors, including microbial invaders. Multipotent intestinal stem cells are essential to epithelial regenerative capacity, generating a programed mixture of absorptive and secretory cell types. Mechanisms of epithelial growth and differentiation in response to endogenous or external stressors remain under investigation. In this review, we highlight the zebrafish, Danio rerio, as a potent model of intestinal epithelial development and function. We describe epithelial composition and key regulators of epithelial renewal to promote the zebrafish as an investigative tool to study epithelial development and growth. We also highlight areas for discovery, particularly in the context of stress-dependent regulation of epithelial function.

Evaluating protective effects of botanicals under inflammation and oxidative stress in chicken apical-out enteroids

Botanicals (BOTs) are well known for their anti-inflammatory and antioxidant activities. They have been widely used as feed additives to reduce inflammation and improve intestinal functions in agricultural animals. However, the effects of BOTs on chicken intestinal epithelial functions are not fully understood. The 3D apical-out chicken enteroids recapitulate the intestinal tissue, and allow convenient access to the luminal surface, thus serving as a suitable model for investigating gut functions. The aim of this study was to identify the roles of BOTs in protecting the intestinal epithelium in chicken enteroids under challenging conditions. Apical-out enteroids were isolated from the small intestines of 18 days-old chicken embryos. Lipopolysaccharide (LPS, 10 µg/mL) and menadione (400 µM) challenges were performed in the media with or without BOTs. Paracellular Fluorescein isothiocyanate-dextran 4kD (FD4) permeability, inflammatory cytokine gene expression, and reactive oxygen species (ROS) generation were analyzed post-BOTs and challenges treatments. Statistical analysis was performed using one-way ANOVA and post hoc multiple comparisons among treatments. The results showed that the LPS challenge for 24 h induced a 50% increase in FD4 permeability compared with nontreated control; thymol, thyme essential oil, and phenol-rich extract significantly (P < 0.02) reduced FD4 permeability by 25%, 41%, and 48% respectively, in comparison with LPS treatment. Moreover, the gene expression of inflammatory cytokines was upregulated, tight junction proteins and defensins were downregulated (P < 0.05) after 6 h of LPS treatment, while these BOTs treatments significantly restored the LPS-induced gene expression alterations (P < 0.05). Menadione oxidative challenge for 1 h significantly increased the ROS level compared with unchallenged control. Enteroids treated with thymol and thyme essential oils showed 30% reduced ROS levels, while the phenol-rich extract reduced them by 60%, in comparison with the challenged group (P < 0.0001). These data confirmed the role of BOTs in supporting the barrier function and reducing the disruptive effects of inflammation and oxidation in the chicken intestine.

The Air-Liquid Interface Reorganizes Membrane Lipids and Enhances the Recruitment of Slc26a3 to Lipid-Rich Domains in Human Colonoid Monolayers.

Cholesterol-rich membrane domains, also called lipid rafts (LRs), are specialized membrane domains that provide a platform for intracellular signal transduction. Membrane proteins often cluster in LRs that further aggregate into larger platform-like structures that are enriched in ceramides and are called ceramide-rich platforms (CRPs). The role of CRPs in the regulation of intestinal epithelial functions remains unknown. Down-regulated in adenoma (DRA) is an intestinal Cl-/HCO3- antiporter that is enriched in LRs. However, little is known regarding the mechanisms involved in the regulation of DRA activity. The air-liquid interface (ALI) was created by removing apical media for a specified number of days; from 12-14 days post-confluency, Caco-2/BBe cells or a colonoid monolayer were grown as submerged cultures. Confocal imaging was used to examine the dimensions of membrane microdomains that contained DRA. DRA expression and activity were enhanced in Caco-2/BBe cells and human colonoids using an ALI culture method. ALI causes an increase in acid sphingomyelinase (ASMase) activity, an enzyme responsible for enhancing ceramide content in the plasma membrane. ALI cultures expressed a larger number of DRA-containing platforms with dimensions >2 µm compared to cells grown as submerged cultures. ASMase inhibitor, desipramine, disrupted CRPs and reduced the ALI-induced increase in DRA expression in the apical membrane. Exposing normal human colonoid monolayers to ALI increased the ASMase activity and enhanced the differentiation of colonoids along with basal and forskolin-stimulated DRA activities. ALI increases DRA activity and expression by increasing ASMase activity and platform formation in Caco-2/BBe cells and by enhancing the differentiation of colonoids.

Lrig1+ small intestinal progenitor cells require MYO5B for proper crypt proliferation and enterocyte maturation

Microvillus inclusion disease (MVID) is a congenital disorder characterized by severe diarrhea. MVID is caused by inactivating mutations in myosin Vb (MYO5B), a motor protein. Mice lacking MYO5B in their intestinal epithelium have demonstrated the importance of MYO5B in enterocyte brush border formation and epithelial cell differentiation. Additionally, the proliferative zone of the intestinal crypt of MYO5B-deficient mice is elongated compared to controls. We hypothesize that the progenitor cell-specific Cre driver under Lrig1 demonstrates crypt-specific effects caused by MYO5B loss. Lrig1-CreERT2;R26R-YFP and Myo5bfl/fl mice were crossbred to generate Lrig1-CreERT2;R26R-YFP;MYO5B fl/fl (Lrig1 ΔMYO5B ) mice. Adult Lrig1 ΔMYO5B mice and control littermates received tamoxifen injection at day 0. GI tissues were collected 2.5, 3, 4, and 5 days after tamoxifen administration for histological characterization. Lrig1 ΔMYO5B mice began experiencing significant weight loss 4 days after tamoxifen injection and lost 20% of their starting body weight on day 5. At day 5, the Lrig1 ΔMYO5B mouse colon was devoid of solid feces and their small intestine contained clear luminal contents, together suggesting a watery diarrheal phenotype. Enterocytes of Lrig1DMYO5B mice on day 5 contained abnormal cytoplasmic accumulation of PAS staining. Fewer goblet cells were apparent in intestinal sections from day 3 and 5 Lrig1 ΔMYO5B mice compared to controls. Crypts were elongated starting at day 3. Similarly, the PCNA+ epithelial region was expanded in Lrig1 ΔMYO5B mice, even reaching into the villi at day 5. The time course of the PCNA+ region expansion corresponded to the wave of MYO5B-loss beginning at the base of crypts. MYO5B expression was diminished in the crypts at day 3 and nearly absent from the epithelium by day 5. Interestingly, expression of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), the rate-limited enzyme of ketogenesis that is important for stem cell function, was expanded in the crypt at day 3 and 5 after tamoxifen. Sodium/glucose cotransporter 1 (SGLT1), which is present along the apical surface of mature enterocytes, was progressively diminished and mis-localized away from the apical surface after tamoxifen administration. Apical sodium-dependent bile acid transporter (ASBT), found in the distal small intestine, demonstrated a similar mis-localization away from the apical membrane in enterocytes.We established a novel mouse model that demonstrates progressive MYO5B loss in the intestinal epithelium, starting at the base of the crypt and reaching villi tips over the course of 5 days following Cre induction. This Lrig1 ΔMYO5B strain will allow for lineage tracing studies to better understand the effect of MYO5B loss on intestinal epithelial differentiation and progenitor function. NSF GFRP, NIH R01DK128190 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.