Reduction of specific enterocytes from loss of intestinal LGR4 improves lipid metabolism in mice.

Whether intestinal Leucine-rich repeat containing G-protein-coupled receptor 4 (LGR4) impacts nutrition absorption and energy homeostasis remains unknown. Here, we report that deficiency of Lgr4 (Lgr4iKO) in intestinal epithelium decreased the proportion of enterocytes selective for long-chain fatty acid absorption, leading to reduction in lipid absorption and subsequent improvement in lipid and glucose metabolism. Single-cell RNA sequencing demonstrates the heterogeneity of absorptive enterocytes, with a decrease in enterocytes selective for long-chain fatty acid-absorption and an increase in enterocytes selective for carbohydrate absorption in Lgr4iKO mice. Activation of Notch signaling and concurrent inhibition of Wnt signaling are observed in the transgenes. Associated with these alterations is the substantial reduction in lipid absorption. Decrement in lipid absorption renders Lgr4iKO mice resistant to high fat diet-induced obesity relevant to wild type littermates. Our study thus suggests that targeting intestinal LGR4 is a potential strategy for the intervention of obesity and liver steatosis.

Analysis of intestinal epithelial cell responses to Cryptosporidium highlights the temporal effects of IFN-γ on parasite restriction.

The production of IFN-γ is crucial for control of multiple enteric infections, but its impact on intestinal epithelial cells (IEC) is not well understood. Cryptosporidium parasites exclusively infect epithelial cells and the ability of interferons to activate the transcription factor STAT1 in IEC is required for parasite clearance. Here, the use of single cell RNA sequencing to profile IEC during infection revealed an increased proportion of mid-villus enterocytes during infection and induction of IFN-γ-dependent gene signatures that was comparable between uninfected and infected cells. These analyses were complemented by in vivo studies, which demonstrated that IEC expression of the IFN-γ receptor was required for parasite control. Unexpectedly, treatment of Ifng-/- mice with IFN-γ showed the IEC response to this cytokine correlates with a delayed reduction in parasite burden but did not affect parasite development. These data sets provide insight into the impact of IFN-γ on IEC and suggest a model in which IFN-γ signalling to uninfected enterocytes is important for control of Cryptosporidium.

Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP4 pathway activation in human colon organoids.

Understanding the mechanisms involved in colonic epithelial differentiation is key to unraveling the alterations causing inflammatory conditions and cancer. Organoid cultures provide an unique tool to address these questions but studies are scarce. We report a differentiation system toward enterocytes and goblet cells, the two major colonic epithelial cell lineages, using colon organoids generated from healthy tissue of colorectal cancer patients. Culture of these organoids in medium lacking stemness agents resulted in a modest ultrastructural differentiation phenotype with low-level expression of enterocyte (KLF4, KRT20, CA1, FABP2) and goblet cell (TFF2, TFF3, AGR2) lineage markers. BMP pathway activation through depletion of Noggin and addition of BMP4 resulted in enterocyte-biased differentiation. Contrarily, blockade of the Notch pathway using the γ-secretase inhibitor dibenzazepine (DBZ) favored goblet cell differentiation. Combination treatment with BMP4 and DBZ caused a balanced strong induction of both lineages. In contrast, colon tumor organoids responded poorly to BMP4 showing only weak signals of cell differentiation, and were unresponsive to DBZ. We also investigated the effects of 1α,25-dihydroxyvitamin D3 (calcitriol) on differentiation. Calcitriol attenuated the effects of BMP4 and DBZ on colon normal organoids, with reduced expression of differentiation genes and phenotype. Consistently, in normal organoids, calcitriol inhibited early signaling by BMP4 as assessed by reduction of the level of phospho-SMAD1/5/8. Our results show that BMP and Notch signaling play key roles in human colon stem cell differentiation to the enterocytic and goblet cell lineages and that calcitriol modulates these processes favoring stemness features.

Insight into the function of voltage-sensing phosphatase in hindgut-derived pseudoplacenta of a viviparous teleost Xenotoca eiseni.

Nutrient absorption is essential for animal survival and development. Our previous study on zebrafish reported that nutrient absorption in lysosome-rich enterocytes (LREs) is promoted by the voltage-sensing phosphatase (VSP), which regulates phosphoinositide (PIP) homeostasis via electrical signaling in biological membranes. However, it remains unknown whether this VSP function is shared by different absorptive tissues in other species. Here, we focused on the function of VSP in a viviparous teleost Xenotoca eiseni, whose intraovarian embryos absorb nutrients from the maternal ovarian fluid through a specialized hindgut-derived pseudoplacental structure called trophotaenia. Xenotoca eiseni VSP (Xe-VSP) is expressed in trophotaenia epithelium, an absorptive tissue functionally similar to zebrafish LREs. Notably, the apical distribution of Xe-VSP in trophotaenia epithelial cells closely resembles zebrafish VSP (Dr-VSP) distribution in zebrafish LREs, suggesting a shared role for VSP in absorptive tissues between the two species. Electrophysiological analysis using a heterologous expression system revealed that Xe-VSP preserves functional voltage sensors and phosphatase activity with the leftward shifted voltage sensitivity compared with zebrafish VSP (Dr-VSP). We also identified a single amino acid variation in the S4 helix of Xe-VSP as one of the factors contributing to the leftward shifted voltage sensitivity. This study highlights the biological variation and significance of VSP in various animal species, as well as hinting at the potential role of VSP in nutrient absorption in X. eiseni trophotaenia.NEW & NOTEWORTHY We investigate the voltage-sensing phosphatase (VSP) in Xenotoca eiseni, a viviparous fish whose intraovarian embryos utilize trophotaenia for nutrient absorption. Although X. eiseni VSP (Xe-VSP) shares key features with known VSPs, its distinct voltage sensitivity arises from species-specific amino acid variation. Xe-VSP in trophotaenia epithelium suggests its involvement in nutrient absorption, similar to VSP in zebrafish enterocytes and potentially in species with similar absorptive cells. Our findings highlight the potential role of VSP across species.

The electroneutral Na+-HCO3- cotransporter NBCn1 (SLC4A7) modulates colonic enterocyte pHi, proliferation, and migration.

NBCn1 (SLC4A7) is one of the two major Na+-HCO3- cotransporters in the human colonic epithelium, expressed predominantly in the highly proliferating colonocytes at the cryptal base. Increased NBCn1 expression levels are reported in tumors, including colorectal cancer. The study explores its importance for maintenance of the intracellular pH (pHi), as well as the proliferative, adhesive, and migratory behavior of the self-differentiating Caco2BBe colonic tumor cell line. In the self-differentiating Caco2BBe cells, NBCn1 mRNA was highly expressed from the proliferative stage until full differentiation. The downregulation of NBCn1 expression by RNA interference affected proliferation and differentiation and decreased intracellular pH (pHi) of the cells in correlation with the degree of knockdown. In addition, a disturbed cell adhesion and reduced migratory speed were associated with NBCn1 knockdown. Murine colonic Nbcn1-/- enteroids also displayed reduced proliferative activity. In the migrating Caco2BBe cells, NBCn1 was found at the leading edge and in colocalization with the focal adhesion markers vinculin and paxillin, which suggests that NBCn1 is involved in the establishment of cell-matrix adhesion. Our data highlight the physiological significance of NBCn1 in modulating epithelial pH homeostasis and cell-matrix interactions in the proliferative region of the colonic epithelium and unravel the molecular mechanism behind pathological overexpression of this transporter in human colorectal cancers.NEW & NOTEWORTHY The transporter NBCn1 plays a central role in maintaining homeostasis within Caco2BBe colonic epithelial cells through its regulation of intracellular pH, matrix adhesion, migration, and proliferation. These observations yield valuable insights into the molecular mechanism of the aberrant upregulation of this transporter in human colorectal cancers.

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.

Lipidomics reveals the significance and mechanism of the cellular ceramide metabolism for rotavirus replication.

As one of the most important causative agents of severe gastroenteritis in children, piglets, and other young animals, species A rotaviruses have adversely impacted both human health and the global swine industry. Vaccines against rotaviruses (RVs) are insufficiently effective, and no specific treatment is available. To understand the relationships between porcine RV (PoRV) infection and enterocytes in terms of the cellular lipid metabolism, we performed an untargeted liquid chromatography mass spectrometry (LC-MS) lipidomics analysis of PoRV-infected IPEC-J2 cells. Herein, a total of 451 lipids (263 upregulated lipids and 188 downregulated lipids), spanning sphingolipid, glycerolipid, and glycerophospholipids, were significantly altered compared with the mock-infected group. Interestingly, almost all the ceramides among these lipids were upregulated during PoRV infection. LC-MS analysis was used to validated the lipidomics data and demonstrated that PoRV replication increased the levels of long-chain ceramides (C16-ceramide, C18-ceramide, and C24-ceramide) in cells. Furthermore, we found that these long-chain ceramides markedly inhibited PoRV infection and that their antiviral actions were exerted in the replication stage of PoRV infection. Moreover, downregulation of endogenous ceramides with the ceramide metabolic inhibitors enhanced PoRV propagation. Increasing the levels of ceramides by the addition of C6-ceramide strikingly suppressed the replication of diverse RV strains. We further found that the treatment with an apoptotic inhibitor could reverse the antiviral activity of ceramide against PoRV replication, demonstrating that ceramide restricted RV infection by inducing apoptosis. Altogether, this study revealed that ceramides played an antiviral role against RV infection, providing potential approaches for the development of antiviral therapies.IMPORTANCERotaviruses (RVs) are among the most important zoonosis viruses, which mainly infected enterocytes of the intestinal epithelium causing diarrhea in children and the young of many mammalian and avian species. Lipids play an essential role in viral infection. A comprehensive understanding of the interaction between RV and lipid metabolism in the enterocytes will be helpful to control RV infection. Here, we mapped changes in enterocyte lipids following porcine RV (PoRV) infection using an untargeted lipidomics approach. We found that PoRV infection altered the metabolism of various lipid species, especially ceramides (derivatives of the sphingosine). We further demonstrated that PoRV infection increased the accumulation of ceramides and that ceramides exerted antiviral effects on RV replication by inducing apoptosis. Our findings fill a gap in understanding the alterations of lipid metabolism in RV-infected enterocytes and highlight the antiviral effects of ceramides on RV infection, suggesting potential approaches to control RV infection.

The Celiac-Disease Superantigen Oligomerizes and Increases Permeability in an Enterocyte Cell Model.

Celiac disease (CeD) is an autoimmune disorder triggered by gluten proteins, affecting approximately 1 % of the global population. The 33-mer deamidated gliadin peptide (DGP) is a metabolically modified wheat-gluten superantigen for CeD. Here, we demonstrate that the 33-mer DGP spontaneously assembles into oligomers with a diameter of approximately 24 nm. The 33-mer DGP oligomers present two main secondary structural motifs-a major polyproline II helix and a minor ß-sheet structure. Importantly, in the presence of 33-mer DGP oligomers, there is a statistically significant increase in the permeability in the gut epithelial cell model Caco-2, accompanied by the redistribution of zonula occludens-1, a master tight junction protein. These findings provide novel molecular and supramolecular insights into the impact of 33-mer DGP in CeD and highlight the relevance of gliadin peptide oligomerization.

Fecal microbiota impacts development of Cryptosporidium parvum in the mouse.

The dependence of Cryptosporidium parasites on host cell metabolites suggests that the development of nutritional interventions to limit parasite proliferation should be feasible. Based on this concept, we are testing dietary interventions to affect the enterocytes' metabolism in a manner that limits intracellular multiplication of the parasite. We hypothesize that changes in the metabolic pathways encoded by the gastro-intestinal tract microbiota may restrict parasite proliferation. To identify taxonomic and metabolic features of the microbiota associated with severity of cryptosporidiosis, as determined by estimating oocyst output, we characterized the fecal microbiota from mice experimentally infected with Cryptosporidium parvum. To eliminate the confounding effect of the interaction between co-housed mice, as well as facilitate the identification of microbiota markers associated with severity of cryptosporidiosis, fecal microbiota from individually caged mice were analyzed. Variation partitioning analysis applied to 16S sequence data from 25 mice belonging to four experiments shows that experiment was by far the biggest source of microbiota variation. Severity of cryptosporidiosis explained a smaller, though significant, fraction of microbiota variation. Notably, this effect was significant in the pre-patent phase of the infection, before mice excreted oocysts. These results are consistent with the pre-patent intestinal microbiota having a modest, but measurable, effect on cryptosporidiosis.

Clinical and public health implications of increasing notifications of LEE-negative Shiga toxin-producing Escherichia coli in England, 2014-2022.

Introduction. Shiga toxin-producing Escherichia coli (STEC) belong to a diverse group of gastrointestinal pathogens. The pathogenic potential of STEC is enhanced by the presence of the pathogenicity island called the Locus of Enterocyte Effacement (LEE), including the intimin encoding gene eae.Gap statement. STEC serotypes O128:H2 (Clonal Complex [CC]25), O91:H14 (CC33), and O146:H21 (CC442) are consistently in the top five STEC serotypes isolated from patients reporting gastrointestinal symptoms in England. However, they are eae/LEE-negative and perceived to be a low risk to public health, and we know little about their microbiology and epidemiology.Aim. We analysed clinical outcomes and genome sequencing data linked to patients infected with LEE-negative STEC belonging to CC25 (O128:H2, O21:H2), CC33 (O91:H14) and, and CC442 (O146:H21, O174:H21) in England to assess the risk to public health.Results. There was an almost ten-fold increase between 2014 and 2022 in the detection of all STEC belonging to CC25, CC33 and CC442 (2014 n=38, 2022 n=336), and a total of 1417 cases. There was a higher proportion of female cases (55-70 %) and more adults than children, with patients aged between 20-40 and >70 most at risk across the different serotypes. Symptoms were consistent across the three dominant serotypes O91:H14 (CC33), O146:H21 (CC442) and O128:H2 (CC25) (diarrhoea >75 %; bloody diarrhoea 25-32 %; abdominal pain 64-72 %; nausea 37-45 %; vomiting 10-24 %; and fever 27-30 %). Phylogenetic analyses revealed multiple events of acquisition and loss of different stx-encoding prophage. Additional putative virulence genes were identified including iha, agn43 and subA.Conclusions. Continued monitoring and surveillance of LEE-negative STEC infections is essential due to the increasing burden of infectious intestinal disease, and the risk that highly pathogenic strains may emerge following acquisition of the Shiga toxin subtypes associated with the most severe clinical outcomes.

Protein Coronas Derived from Mucus Act as Both Spear and Shield to Regulate Transferrin Functionalized Nanoparticle Transcellular Transport in Enterocytes.

The epithelial mucosa is a key biological barrier faced by gastrointestinal, intraoral, intranasal, ocular, and vaginal drug delivery. Ligand-modified nanoparticles demonstrate excellent ability on this process, but their efficacy is diminished by the formation of protein coronas (PCs) when they interact with biological matrices. PCs are broadly implicated in affecting the fate of NPs in vivo and in vitro, yet few studies have investigated PCs formed during interactions of NPs with the epithelial mucosa, especially mucus. In this study, we constructed transferrin modified NPs (Tf-NPs) as a model and explored the mechanisms and effects that epithelial mucosa had on PCs formation and the subsequent impact on the transcellular transport of Tf-NPs. In mucus-secreting cells, Tf-NPs adsorbed more proteins from the mucus layers, which masked, displaced, and dampened the active targeting effects of Tf-NPs, thereby weakening endocytosis and transcellular transport efficiencies. In mucus-free cells, Tf-NPs adsorbed more proteins during intracellular trafficking, which enhanced transcytosis related functions. Inspired by soft coronas and artificial biomimetic membranes, we used mucin as an "active PC" to precoat Tf-NPs (M@Tf-NPs), which limited the negative impacts of "passive PCs" formed during interface with the epithelial mucosa and improved favorable routes of endocytosis. M@Tf-NPs adsorbed more proteins associated with endoplasmic reticulum-Golgi functions, prompting enhanced intracellular transport and exocytosis. In summary, mucus shielded against the absorption of Tf-NPs, but also could be employed as a spear to break through the epithelial mucosa barrier. These findings offer a theoretical foundation and design platform to enhance the efficiency of oral-administered nanomedicines.

Aichivirus A1 replicates in human intestinal epithelium and bronchial tissue: Lung-gut axis?

The role of aichivirus A1 (AiV-A1) in acute gastroenteritis remains controversial and in vitro data illustrating its pathogenesis in suitable human models are scarce. Here, we demonstrate that AiV-A1 isolate A846/88 replicates in ApoA1- (absorptive) and Ki-67-positive (proliferative) enterocytes in stem cell-derived human small intestinal epithelium (HIE) as well as in patient biopsy samples, but not in any of the tested human cell lines. The infection did not result in tissue damage and did not trigger type I and type III interferon (IFN) signalling, whereas the control, human coxsackievirus B3 (strain Nancy), triggered both IFNs. To investigate the tissue tropism, we infected a human tracheal/bronchial epithelium model (HTBE) with AiV-A1 isolates A846/88 and kvgh99012632/2010 and, as a control, with rhinovirus A2 (RV-A2). AiV-A1 isolate kvgh99012632/2010, but not isolate A846/88, replicated in HTBE and induced type III IFN and ISGs signalling. By using various pharmacological inhibitors, we elaborated that cellular entry of AiV-A1 depends on clathrin, dynamin, and lipid rafts and is strongly reliant on endosome acidification. Viral particles co-localised with Rab5a-positive endosomes and promoted leakage of endosomal content. Our data shed light on the early events of AiV-A1 infection and reveal that different isolates exhibit distinct tissue tropism. This supports its clinical importance as a human pathogen with the potential to evolve toward broader tissue specificity.

Bicarbonate secretion and acid/base sensing by the intestine.

The transport of bicarbonate across the enterocyte cell membrane regulates the intracellular as well as the luminal pH and is an essential part of directional fluid movement in the gut. Since the first description of "active" transport of HCO3- ions against a concentration gradient in the 1970s, the fundamental role of HCO3- transport for multiple intestinal functions has been recognized. The ion transport proteins have been identified and molecularly characterized, and knockout mouse models have given insight into their individual role in a variety of functions. This review describes the progress made in the last decade regarding novel techniques and new findings in the molecular regulation of intestinal HCO3- transport in the different segments of the gut. We discuss human diseases with defects in intestinal HCO3- secretion and potential treatment strategies to increase luminal alkalinity. In the last part of the review, the cellular and organismal mechanisms for acid/base sensing in the intestinal tract are highlighted.

Lizhong decoction ameliorates ulcerative colitis by inhibiting ferroptosis of enterocytes via the Nrf2/SLC7A11/GPX4 pathway.

ETHNOPHARMACOLOGY RELEVANCE: Traditional herbal medicines have been considered as a novel and effective way to treat many diseases. Lizhong decoction (LZD), a classical prescription composed of Zingiber officinale Rosc., Panax ginseng C. A. Mey., Atractylodes macrocephala Koidz., and Glycyrrhiza uralensis Fisch., has been used to treat gastrointestinal disorders in clinical practices for thousands of years. However, the mechanism of LZD in alleviating ulcerative colitis (UC) is still unclear. AIM OF THE STUDY: The purpose of this study was to clarify the potential molecular mechanism of LZD in improving UC. MATERIALS AND METHODS: The amelioration of LZD on dextran sodium sulfate (DSS)-induced UC mice was evaluated by body weight, colon length, pathology of colon tissues, pro-inflammatory cytokines, and intestinal tight junction (TJ) proteins. Moreover, the gene expression profiles of UC patients were extracted to investigate potential pathological mechanisms of UC. The influence of LZD on ferroptosis was analyzed by iron load, malondialdehyde (MDA), and the expression of ferroptosis-associated proteins. Meanwhile, the inhibition of LZD on oxidative stress (OS) was assessed by the superoxide dismutase (SOD) activity, as well as the expression levels of glutathione (GSH) and glutathione disulfide (GSSG). Furthermore, the influence of LZD on ferroptosis was assessed by inhibiting nuclear factor (erythroid-derived-2)-like 2 (Nrf2). RESULTS: LZD showed significant therapeutic effects in UC mice, including reduction of intestinal injury and inflammation. Moreover, LZD treatment notably upregulated the expression of TJ proteins. Further investigation indicated that LZD significantly inhibited the ferroptosis of enterocytes by decreasing iron load and MDA, and increasing the expression levels of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) in colon tissues. Furthermore, the decreased activity of SOD, reduced level of GSH, and increased content of GSSG in UC mice were notably reversed by LZD. Consistent with in vivo results, LZD could markedly inhibit ferroptosis and OS in RSL3-induced Caco-2 cells. Mechanistically, LZD alleviated ferroptosis by suppressing OS through the activation of Nrf2 signaling. CONCLUSIONS: Collectively, LZD remarkably improved intestinal pathological injury in UC mice, and its potential mechanism was the suppression of ferroptosis in enterocytes by the Nrf2/SLC7A11/GPX4 pathway.

Drosophila activins adapt gut size to food intake and promote regenerative growth.

Rapidly renewable tissues adapt different strategies to cope with environmental insults. While tissue repair is associated with increased intestinal stem cell (ISC) proliferation and accelerated tissue turnover rates, reduced calorie intake triggers a homeostasis-breaking process causing adaptive resizing of the gut. Here we show that activins are key drivers of both adaptive and regenerative growth. Activin-ß (Actß) is produced by stem and progenitor cells in response to intestinal infections and stimulates ISC proliferation and turnover rates to promote tissue repair. Dawdle (Daw), a divergent Drosophila activin, signals through its receptor, Baboon, in progenitor cells to promote their maturation into enterocytes (ECs). Daw is dynamically regulated during starvation-refeeding cycles, where it couples nutrient intake with progenitor maturation and adaptive resizing of the gut. Our results highlight an activin-dependent mechanism coupling nutrient intake with progenitor-to-EC maturation to promote adaptive resizing of the gut and further establish activins as key regulators of adult tissue plasticity.

Low dietary carbohydrate induces structural alterations in enterocytes of the chicken ileum.

We investigated the role of dietary carbohydrates in the maintenance of the enterocyte microvillar structure in the chicken ileum. Male chickens were divided into the control and three experimental groups, and the experimental groups were fed diets containing 50%, 25%, and 0% carbohydrates of the control diet. The structural alterations in enterocytes were examined using transmission electron microscopy and immunofluorescent techniques for ß-actin and villin. Glucagon-like peptide (GLP)-2 and proglucagon mRNA were detected by immunohistochemistry and in situ hybridization, respectively. Fragmentation and wide gap spaces were frequently observed in the microvilli of the 25% and 0% groups. The length, width, and density of microvilli were also decreased in the experimental groups. The experimental groups had shorter terminal web extensions, and there were substantial changes in the mitochondrial density between the control and experimental groups. Intensities of ß-actin and villin immunofluorescence observed on the apical surface of enterocytes were lower in the 0% group. The frequency of GLP-2-immunoreactive and proglucagon mRNA-expressing cells decreased with declining dietary carbohydrate levels. This study revealed that dietary carbohydrates contribute to the structural maintenance of enterocyte microvilli in the chicken ileum. The data from immunohistochemistry and in situ hybridization assays suggest the participation of GLP-2 in this maintenance system.

Reduced surface pH and upregulated AE2 anion exchange in SLC26A3-deleted polarized intestinal epithelial cells.

Loss of function mutations in the SLC26A3 gene cause chloride-losing diarrhea in mice and humans. Although systemic adaptive changes have been documented in these patients and in the corresponding knockout mice, how colonic enterocytes adapt to loss of this highly expressed and highly regulated luminal membrane anion exchanger remains unclear. To address this question, SLC26A3 was deleted in the self-differentiating Caco2BBe colonic cell line by the CRISPR/Cas9 technique. We selected a clone with loss of SLC26A3 protein expression and morphological features indistinguishable from those of the native cell line. Neither growth curves nor development of transepithelial electrical resistance (TEER) differed between wild-type (WT) and SLC26A3 knockout (KO) cells. Real-time qPCR and Western analysis in SLC26A3-KO cells revealed an increase in AE2 expression without significant change in NHE3 expression or localization. Steady-state pHi and apical and basolateral Cl-/HCO3- exchange activities were assessed fluorometrically in a dual perfusion chamber with independent perfusion of luminal and serosal baths. Apical Cl-/HCO3- exchange rates were strongly reduced in SLC26A3-KO cells, accompanied by a surface pH more acidic than that of WT cells. Steady-state pHi was not significantly different from that of WT cells, but basolateral Cl-/HCO3- exchange rates were higher in SLC26A3-KO than in WT cells. The data show that CRISPR/Cas9-mediated SLC26A3 deletion strongly reduced apical Cl-/HCO3- exchange rate and apical surface pH, but sustained a normal steady-state pHi due to increased expression and function of basolateral AE2. The low apical surface pH resulted in functional inhibition of NHE-mediated fluid absorption despite normal expression of NHE3 polypeptide.NEW & NOTEWORTHY SLC26A3 gene mutations cause chloride-losing diarrhea. To understand how colonic enterocytes adapt, SLC26A3 was deleted in Caco2BBe cells using CRISPR/Cas9. In comparison to the wild-type cells, SLC26A3 knockout cells showed similar growth and transepithelial resistance but substantially reduced apical Cl-/HCO3- exchange rates, and an acidic surface pH. Steady-state intracellular pH was comparable between the WT and KO cells due to increased basolateral AE2 expression and function.

New Maintenance Culture Method for Intestinal Stem Cells Derived from Human Induced Pluripotent Stem Cells.

Most orally administered drugs exert their effects after being absorbed in the small intestine. Therefore, new drugs must undergo nonclinical pharmacokinetic evaluations in the small intestine. Enterocytes derived from human induced pluripotent stem cells (hiPSCs) are expected to be used in the evaluation system, as they reflect human intestinal characteristics more accurately; moreover, several differentiation protocols are available for these cells. However, enterocytes derived from hiPSCs have drawbacks such as time, cost, and lot-to-lot differences. Hence, to address these issues, we attempted to maintain hiPSC-derived intestinal stem cells (ISCs) that can differentiate into various intestinal cells by regulating various pathways. Although our previous attempt was partly successful, the drawbacks of elevated cost and complicated handling remained, because more than 10 factors (A 83-01, CHIR99021, epidermal growth factor, basic fibroblast growth factor, SB202190, nicotinamide, N-acetylcysteine, valproic acid, Wnt3a, R-spondin 1, and noggin) are needed to maintain ISCs. Therefore, in this study, we successfully maintained ISCs using only five factors, including growth factors. Moreover, we generated not only enterocytes but also intestinal organoids from the maintained ISCs. Thus, our novel findings provided a time-saving and cost-effective culture method for enterocytes derived from hiPSCs.

Mitochondrial dysfunction abrogates dietary lipid processing in enterocytes.

Digested dietary fats are taken up by enterocytes where they are assembled into pre-chylomicrons in the endoplasmic reticulum followed by transport to the Golgi for maturation and subsequent secretion to the circulation1. The role of mitochondria in dietary lipid processing is unclear. Here we show that mitochondrial dysfunction in enterocytes inhibits chylomicron production and the transport of dietary lipids to peripheral organs. Mice with specific ablation of the mitochondrial aspartyl-tRNA synthetase DARS2 (ref. 2), the respiratory chain subunit SDHA3 or the assembly factor COX10 (ref. 4) in intestinal epithelial cells showed accumulation of large lipid droplets (LDs) in enterocytes of the proximal small intestine and failed to thrive. Feeding a fat-free diet suppressed the build-up of LDs in DARS2-deficient enterocytes, which shows that the accumulating lipids derive mostly from digested fat. Furthermore, metabolic tracing studies revealed an impaired transport of dietary lipids to peripheral organs in mice lacking DARS2 in intestinal epithelial cells. DARS2 deficiency caused a distinct lack of mature chylomicrons concomitant with a progressive dispersal of the Golgi apparatus in proximal enterocytes. This finding suggests that mitochondrial dysfunction results in impaired trafficking of chylomicrons from the endoplasmic reticulum to the Golgi, which in turn leads to storage of dietary lipids in large cytoplasmic LDs. Taken together, these results reveal a role for mitochondria in dietary lipid transport in enterocytes, which might be relevant for understanding the intestinal defects observed in patients with mitochondrial disorders5.

Correlates of Plasma Citrulline, a Potential Marker of Enterocyte Mass, among Children with Stunting: A Cross-Sectional Study in Uganda.

BACKGROUND: Environmental enteric dysfunction (EED) is associated with stunting. Citrulline, produced in mature enterocytes, may be a valuable biomarker of small intestinal enterocyte mass in the context of EED. OBJECTIVES: We aimed to explore the correlates of plasma citrulline (p-cit) in children with stunting. METHODS: In a cross-sectional study using baseline data from the community-based MAGNUS (milk affecting growth, cognition and the gut in child stunting) trial (ISRCTN13093195), we explored potential correlates of p-cit in Ugandan children with stunting aged 12-59 mo. Using linear regression in univariate and multivariate models, we explored associations with socioeconomics, diet, micronutrient status, and water, sanitation, and hygiene characteristics. The influence of covariates age, fasting, and systemic inflammation were also explored. RESULTS: In 750 children, the mean ± standard deviation age was 32.0 ± 11.7 mo, and height-for-age z-score was -3.02 ± 0.74. P-cit, available for 730 children, differed according to time fasted and was 20.7 ± 8.9, 22.3 ± 10.6 and 24.2 ± 13.1 µmol/L if fasted <2, 2-5 and >5 h, respectively. Positive correlates of p-cit were age [0.07; 95% confidence interval (CI): 0.001, 0.15 µmol/L] and log10 serum insulin-like growth factor-1 (8.88; 95% CI: 5.09, 12.67 µmol/L). With adjustment for systemic inflammation, the association with serum insulin-like growth factor-1 reduced (4.98; 95% CI: 0.94, 9.03 µmol/L). Negative correlates of p-cit included food insecurity, wet season (-3.12; 95% CI: -4.97, -1.26 µmol/L), serum C-reactive protein (-0.15; 95% CI: -0.20, -0.10 µmol/L), serum α1-acid glycoprotein (-5.34; 95% CI: -6.98, -3.70 µmol/L) and anemia (-1.95; 95% CI: -3.72, -0.18 µmol/L). Among the negatively correlated water, sanitation, and hygiene characteristics was lack of soap for handwashing (-2.53; 95% CI: -4.82, -0.25 µmol/L). Many associations attenuated with adjustment for inflammation. CONCLUSIONS: Many of the correlates of p-cit are characteristic of populations with a high EED prevalence. Systemic inflammation is strongly associated with p-cit and is implicated in EED and stunting. Adjustment for systemic inflammation attenuates many associations, reflecting either confounding, mediation, or both. This study highlights the complex interplay between p-cit and systemic inflammation.