Effects of exposure to the neonicotinoid pesticide clothianidin on α-defensin secretion and gut microbiota in mice.

The mechanism by which the neonicotinoid pesticide clothianidin (CLO) disrupts the intestinal microbiota of experimental animals is unknown. We focused on α-defensins, which are regulators of the intestinal microbiota. Subchronic exposure to CLO induced dysbiosis and reduced short-chain fatty acid-producing bacteria in the intestinal microbiota of mice. Levels of cryptdin-1 (Crp1, a major α-defensin in mice) in feces and cecal contents were lower in the CLO-exposed groups than in control. In Crp1 immunostaining, Paneth cells in the jejunum and ileum of the no-observed-adverse-effect-level CLO-exposed group showed a stronger positive signal than control, likely due to the suppression of Crp1 release. Our results showed that CLO exposure suppresses α-defensin secretion from Paneth cells as part of the mechanism underlying CLO-induced dysbiosis.

A Basic Study of the Effects of Mulberry Leaf Administration to Healthy C57BL/6 Mice on Gut Microbiota and Metabolites.

Mulberry leaves contain α-glucosidase inhibitors, which have hypoglycemic effects and are considered functional foods. However, few reports have covered the effects of mulberry leaf components on normal gut microbiota and gut metabolites. Herein, gut microbiota analysis and NMR-based metabolomics were performed on the feces of mulberry leaf powder (MLP)-treated mice to determine the effects of long-term MLP consumption. Gut microbiota in the mouse were analyzed using 16S-rRNA gene sequencing, and no significant differences were revealed in the diversity and community structure of the gut microbiota in the C57BL/6 mice with or without MLP supplementation. Thirty-nine metabolites were identified via 1H-NMR analysis, and carbohydrates and amino acids were significantly (p < 0.01-0.05) altered upon MLP treatment. In the MLP-treated group, there was a marked increase and decrease in maltose and glucose concentrations, respectively, possibly due to the degradation inhibitory activity of oligosaccharides. After 5 weeks, all amino acid concentrations decreased. Furthermore, despite clear fluctuations in fecal saccharide concentrations, short-chain fatty acid production via intestinal bacterial metabolism was not strongly affected. This study provides the knowledge that MLP administration can alter the gut metabolites without affecting the normal gut microbiota, which is useful for considering MLP as a healthy food source.

Antimicrobial Properties and Mode of Action of Cryptdin-4, a Mouse α-Defensin Regulated by Peptide Redox Structures and Bacterial Cultivation Conditions.

Cryptdin-4 (crp4) is an enteric α-defensin derived from mice, and is a main mediator of immunity to oral infections and a determinant of the composition of the intestinal microbiota. Structurally, crp4 exists in two states: the oxidized form (crp4oxi), constrained by three invariant disulfide bonds, and the reduced form (crp4red) with six free thiol groups, both of which exist in the intestinal tract. In this study, the antibacterial mechanisms of crp4 in both forms under aerobic and anaerobic conditions were investigated using Escherichia coli (E. coli), an anaerobic facultative bacterium, as a model. Fluorescent dye studies revealed that both crp4oxi and crp4red exhibited antimicrobial activity against cells cultured under aerobic conditions via rapid membrane depolarization. Furthermore, the antioxidant treatment experiments suggested that only crp4oxi exhibited antimicrobial activity by the induction and accumulation of reactive oxygen species (ROS). However, under anaerobic culture conditions, the ability of both forms to disrupt the function of bacterial membranes decreased and activity was greatly reduced, but crp4red maintained some antimicrobial activity. This activity may be due to the inhibition of intracellular functions by DNA binding. Altogether, these data indicate that, according to its redox structure and the environmental redox conditions, crp4 could perform different antimicrobial activities via different mechanisms.

Application of Benchtop NMR for Metabolomics Study Using Feces of Mice with DSS-Induced Colitis.

Nuclear magnetic resonance (NMR)-based metabolomics, which comprehensively measures metabolites in biological systems and investigates their response to various perturbations, is widely used in research to identify biomarkers and investigate the pathogenesis of underlying diseases. However, further applications of high-field superconducting NMR for medical purposes and field research are restricted by its high cost and low accessibility. In this study, we applied a low-field, benchtop NMR spectrometer (60 MHz) employing a permanent magnet to characterize the alterations in the metabolic profile of fecal extracts obtained from dextran sodium sulfate (DSS)-induced ulcerative colitis model mice and compared them with the data acquired from high-field NMR (800 MHz). Nineteen metabolites were assigned to the 60 MHz 1H NMR spectra. Non-targeted multivariate analysis successfully discriminated the DSS-induced group from the healthy control group and showed high comparability with high-field NMR. In addition, the concentration of acetate, identified as a metabolite with characteristic behavior, could be accurately quantified using a generalized Lorentzian curve fitting method based on the 60 MHz NMR spectra.

Decreased Paneth cell α-defensins promote fibrosis in a choline-deficient L-amino acid-defined high-fat diet-induced mouse model of nonalcoholic steatohepatitis via disrupting intestinal microbiota.

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fibrosis that develops from fatty liver. Disruption of intestinal microbiota homeostasis, dysbiosis, is associated with fibrosis development in NASH. An antimicrobial peptide α-defensin secreted by Paneth cells in the small intestine is known to regulate composition of the intestinal microbiota. However, involvement of α-defensin in NASH remains unknown. Here, we show that in diet-induced NASH model mice, decrease of fecal α-defensin along with dysbiosis occurs before NASH onset. When α-defensin levels in the intestinal lumen are restored by intravenous administration of R-Spondin1 to induce Paneth cell regeneration or by oral administration of α-defensins, liver fibrosis is ameliorated with dissolving dysbiosis. Furthermore, R-Spondin1 and α-defensin improved liver pathologies together with different features in the intestinal microbiota. These results indicate that decreased α-defensin secretion induces liver fibrosis through dysbiosis, further suggesting Paneth cell α-defensin as a potential therapeutic target for NASH.

Shorter sleep time relates to lower human defensin 5 secretion and compositional disturbance of the intestinal microbiota accompanied by decreased short-chain fatty acid production.

Sleep is essential for our health. Short sleep is known to increase disease risks via imbalance of intestinal microbiota, dysbiosis. However, mechanisms by which short sleep induces dysbiosis remain unknown. Small intestinal Paneth cell regulates the intestinal microbiota by secreting antimicrobial peptides including α-defensin, human defensin 5 (HD5). Disruption of circadian rhythm mediating sleep-wake cycle induces Paneth cell failure. We aim to clarify effects of short sleep on HD5 secretion and the intestinal microbiota. Fecal samples and self-reported sleep time were obtained from 35 healthy middle-aged Japanese (41 to 60-year-old). Shorter sleep time was associated with lower fecal HD5 concentration (r = 0.354, p = 0.037), lower centered log ratio (CLR)-transformed abundance of short-chain fatty acid (SCFA) producers in the intestinal microbiota such as [Ruminococcus] gnavus group (r = 0.504, p = 0.002) and Butyricicoccus (r = 0.484, p = 0.003), and lower fecal SCFA concentration. Furthermore, fecal HD5 positively correlated with the abundance of these genera and SCFA concentration. These findings suggest that short sleep relates to disturbance of the intestinal microbiota via decreased HD5 secretion.

Associations between Maternal Diet, Human Milk Macronutrients, and Breast-Fed Infant Growth during the First Month of Life in the SMILE Iwamizawa in Japan.

Maternal diet may affect human milk macronutrients, but it remains to be elucidated whether this is also influential in infant growth. This study aimed to examine (1) how maternal diet influences human milk macronutrients, and (2) to what extent the variation in milk macronutrients affects infant growth during the first month of life. In 71 Japanese lactating women, maternal dietary information was collected from the brief-type self-administered diet history questionnaire, and anthropometry of mother-infant dyads was collected from medical records. Macronutrients in milk were analyzed by a Human Milk Analyzer. Maternal retinol intake was associated with the carbohydrate content in human milk at 1-month postpartum (standardized ß coefficient: 0.287; p = 0.038). Moreover, the energy content in human milk was associated with an increase in the weight standard deviation score based on the WHO growth standard at 1 month of age (standardized ß coefficient: 0.399; p = 0.046). Nevertheless, the milk macronutrient was not associated with the risk of infant growth abnormalities. In conclusion, a part of the maternal diet impacts macronutrient contents in human milk, but milk macronutrients have a limited effect on infant growth only within the normal growth curve during the first month of life.

Efficient recombinant production of mouse-derived cryptdin family peptides by a novel facilitation strategy for inclusion body formation.

BACKGROUND: A number of antimicrobial peptides (AMPs) hold promise as new drugs owing to their potent bactericidal activity and because they are often refractory to the development of drug resistance. Cryptdins (Crps) are a family of antimicrobial peptides found in the small intestine of mice, comprising six isoforms containing three sets of disulfide bonds. Although Crp4 is actively being investigated, there have been few studies to date on the other Crp isoforms. A prerequisite for detailed characterization of the other Crp isoforms is establishment of efficient sample preparation methods. RESULTS: To avoid degradation during recombinant expression of Crps in E. coli, co-expression of Crps with the aggregation-prone protein human α-lactalbumin (HLA) was used to promote the formation of stable inclusion bodies. Using this method, the production of Crp4 and Crp6 by the BL21 strain was effective, but the expression of other Crp isoforms was not as efficient. The results of a cell-free system study suggested that Crps were degraded, even though a substantial amounts of Crps were synthesized. Therefore, using the Origami™ B strain, we were able to significantly increase the expression efficiency of Crps by promoting the formation of erroneous intermolecular disulfide bonds between HLA and Crps, thereby promoting protein aggregation and inclusion body formation, which prevented degradation. The various Crp isoforms were successfully refolded in vitro and purified using reversed-phase HPLC. In addition, the yield was further improved by deformylation of formyl-Crps. We measured the antibacterial activity of Crps against both Gram-positive and Gram-negative bacteria. Each Crp isoform exhibited a completely different trend in antimicrobial activity, although conformational analysis by circular dichroism did not reveal any significant steric differences. CONCLUSION: In this study, we established a novel and efficient method for the production of the cryptdin family of cysteine-containing antimicrobial peptides. Additionally, we found that there were notable differences in the antibacterial activities of the various Crp family members. The expression system established in this study is expected to provide new insights regarding the mechanisms underlying the different antibacterial activities of the Crp family of peptides.

Reactive granulopoiesis depends on T-cell production of IL-17A and neutropenia-associated alteration of gut microbiota.

Granulopoiesis in the bone marrow adjusts cellular output as demand for neutrophils changes. Reactive granulopoiesis is induced by profound neutropenia, but its mechanism remains to be clarified. We herein explored its mechanisms using mouse models of syngeneic hematopoietic stem cell transplantation (SCT) and 5-fluorouracil-induced neutropenia. After SCT, T cell production of IL-17A was up-regulated. Neutrophil recovery was significantly delayed in IL-17A-deficient or T cell-deficient RAG1-/- mice, and adoptive transfer of wild-type (WT) T cells facilitated neutrophil engraftment. Gut decontamination with oral antibiotics suppressed T cell production of IL-17A and impaired neutrophil recovery. Transplantation of fecal microbiota collected from neutropenic, not naive, mice promoted neutrophil recovery in these mice, suggesting that neutropenia-associated microbiota had a potential to stimulate reactive granulopoiesis. Our study uncovered a cross talk between gut microbiota and neutropenia after SCT and chemotherapy.

Efficient and simple genetic engineering of enteroids using mouse isolated crypts for investigating intestinal functions.

Intestinal epithelial cells separate subepithelial tissues from luminal environment formed with food, incoming pathogens, and resident intestinal microbiota, etc., and elicit various intestinal function. Enteroid, a three-dimensional culture system of small intestinal epithelial cells, has been widely used for analyzing the intestinal function, further a transgenic enteroid was developed to investigate the molecular mechanisms. However, conventional transgenic enteroid production method, which transfer gene into single stem cells, has limitations including low efficiency and time-consuming. Here we show that by gene transfer into small intestinal isolated crypts maintaining stem cell niche, a transgenic enteroid was obtained quickly and efficiently. Isolated crypts were transfected by lentiviral vector without separating into single cells, and transgenic enteroid composed of all lineages of intestinal epithelial cells was generated at day 7 with yield of 56%, maintaining the intestinal function in drug transport and innate immunity. Our efficient and simple transgenic enteroid generation method enables high-throughput investigation of intestinal epithelial cells and contributes to understanding intestinal function.

Hemodynamic response to intestinal pH stimulation measured with spectroscopic video imaging.

To investigate the relationship between the gut and skin (gut-skin axis), head skin hemodynamic responses to gut stimulation including the injection of acetic acid in nude mice were measured by spectroscopic video imaging, which was calculated using a modified Beer-Lambert formula. The relationship with blood proteins was also analyzed. The blood volume changes in three mice injected with acetic acid were highly reproducible in the mathematical model equation. Four proteins correlated with blood volume changes were all related to immunity. These results suggest that intestinal pH can alter the blood volume in the skin and induce immune-related responses.

Intestinal commensal microbiota and cytokines regulate Fut2+ Paneth cells for gut defense.

Paneth cells are intestinal epithelial cells that release antimicrobial peptides, such as α-defensin as part of host defense. Together with mesenchymal cells, Paneth cells provide niche factors for epithelial stem cell homeostasis. Here, we report two subtypes of murine Paneth cells, differentiated by their production and utilization of fucosyltransferase 2 (Fut2), which regulates α(1,2)fucosylation to create cohabitation niches for commensal bacteria and prevent invasion of the intestine by pathogenic bacteria. The majority of Fut2- Paneth cells were localized in the duodenum, whereas the majority of Fut2+ Paneth cells were in the ileum. Fut2+ Paneth cells showed higher granularity and structural complexity than did Fut2- Paneth cells, suggesting that Fut2+ Paneth cells are involved in host defense. Signaling by the commensal bacteria, together with interleukin 22 (IL-22), induced the development of Fut2+ Paneth cells. IL-22 was found to affect the α-defensin secretion system via modulation of Fut2 expression, and IL-17a was found to increase the production of α-defensin in the intestinal tract. Thus, these intestinal cytokines regulate the development and function of Fut2+ Paneth cells as part of gut defense.

Ingestion of miso regulates immunological robustness in mice.

In Japan, there is a long history of consumption of miso, a fermented soybean paste, which possesses beneficial effects on human health. However, the mechanism behind these effects is not fully understood. To clarify the effects of miso on immune cells, we evaluated its immunomodulatory activity in mice. Miso did not alter the percentage of B and T cells in the spleen; however, it increased CD69+ B cells, germinal center B cells and regulatory T cells. Anti-DNA immunoglobulin M antibodies, which prevent autoimmune disease, were increased following ingestion of miso. Transcriptome analysis of mouse spleen cells cultured with miso and its raw material revealed that the expression of genes, including interleukin (IL)-10, IL-22 and CD86, was upregulated. Furthermore, intravital imaging of the small intestinal epithelium using a calcium biosensor mouse line indicated that miso induced Ca2+ signaling in a manner similar to that of probiotics. Thus, ingestion of miso strengthened the immune response and tolerance in mice. These results appear to account, at least in part, to the salubrious effects of miso.

Oral frailty and carriage of oral Candida in community-dwelling older adults (Check-up to discover Health with Energy for senior Residents in Iwamizawa; CHEER Iwamizawa).

OBJECTIVE: To examine the association between oral frailty and oral Candida carriage as a general indicator of deteriorating oral function in older adults. BACKGROUND: Older adults exhibit an elevated risk of oral candidiasis caused by Candida. Although many studies have identified factors associated with oral Candida carriage, none have evaluated its relationship with oral function. MATERIALS AND METHODS: This study included 210 community-dwelling older adults aged ≥60 years who participated in wellness checks. Fungal flora expression in saliva samples was evaluated to identify oral C. albicans and C. glabrata. Participants were categorised by detection of neither strain (group 1), either one of the strains (group 2), or both strains (group 3). The relationship between oral Candida carriage and oral frailty was evaluated by multinomial logistic regression analysis. RESULTS: The participants included 58 men and 152 women with a mean age of 74.2 ± 6.1 years. A total of 88 (41.9%), 94 (44.8%) and 28 (13.3%) participants were assigned to groups 1, 2 and 3 respectively. In the multinomial logistic regression analysis, significant associations were observed between group 1 and group 2 for "Have you choked on your tea or soup recently?" and the number of applicable oral frailty items. Between group 1 and group 3, significant associations were observed for the number of remaining teeth, masticatory performance and the number of applicable oral frailty items. CONCLUSION: We obtained basic data useful for intervention studies aimed at verifying whether oral function management prevents deterioration of the oral bacterial flora.

Potent bactericidal activity of reduced cryptdin-4 derived from its hydrophobicity and mediated by bacterial membrane disruption.

Defensin is a cysteine-rich antimicrobial peptide with three disulphide bonds under normal oxidative conditions. Cryptdin-4 (Crp4) is a defensin secreted by Paneth cells in the small intestine of mice, and only reduced Crp4 (Crp4red) shows activity against enteric commensal bacteria, although both oxidised Crp4 (Crp4ox) and Crp4red can kill non-commensal bacteria. To investigate the molecular factors that affect the potent antimicrobial activity of Crp4red, the bactericidal activities of Crp4ox and Crp4red, Crp4 with all Cys residues substituted with Ser peptide (6C/S-Crp4), and Crp4 with all thiol groups modified by N-ethylmaleimide (NEM-Crp4) were assessed. All peptides showed bactericidal activity against non-commensal bacteria, whereas Crp4red and NEM-Crp4 showed bactericidal activity against commensal bacteria. These potent peptides exhibited high hydrophobicity, which was strongly correlated with membrane insertion. Intriguingly, Crp4ox formed electrostatic interactions with the membrane surface of bacteria, even without exerting bactericidal activity. Moreover, the bactericidal activity of both oxidised and reduced forms of Crp4 was abolished by inhibition of electrostatic interactions; this finding suggests that Crp4red targets bacterial membranes. Finally, a liposome leakage assay against lipids extracted from commensal bacteria demonstrated a correlation with bactericidal activity. These results suggest that the potent bactericidal activity of Crp4red is derived from its hydrophobicity, and the bactericidal mechanism involves disruption of the bacterial membrane. Findings from this study provide a better understanding of the bactericidal mechanism of both Crp4ox and Crp4red.

Lower human defensin 5 in elderly people compared to middle-aged is associated with differences in the intestinal microbiota composition: the DOSANCO Health Study.

Recently, aging is considered a risk factor for various diseases. Although changes in the intestinal microbiota along with aging are thought to associate with the increased disease risk, mechanisms that cause age-related transition of the intestinal microbiota remain unknown. This study aims to clarify relationships between the amount of human defensin 5 (HD5), a Paneth cell α-defensin, which is known to regulate the intestinal microbiota, and age-related differences of the intestinal microbiota composition. Fecal samples from 196 healthy Japanese (35 to 81 years old) were collected and measured HD5 concentration. HD5 concentration in the elderly group (age > 70 years old) was significantly lower than the middle-aged group (age ≤ 70 years old). Furthermore, individual age was negatively correlated with HD5 concentration (r = - 0.307, p < 0.001). In ß-diversity, the intestinal microbiota of the elderly showed a significantly different composition compared to the middle-aged. At the genus level, relative abundance of Collinsella, Alistipes, Peptococcaceae; unassigned, Lactobacillus, Lactococcus, Weissella, Christensenellaceae R-7 group, Megasphaera, and [Eubacterium] eligens group was significantly higher, and Lachnospiraceae; unassigned, Blautia, Anaerostipes, Fusicatenibacter, Dorea, and Faecalibacterium was significantly lower in the elderly compared to the middle-aged. In addition, HD5 concentration was negatively correlated with Alistipes, Peptococcaceae; unassigned, and Christensenellaceae R-7 group and positively correlated with Lachnospiraceae; unassigned and Dorea. These results provide novel insights into the immunosenescence of enteric innate immunity, indicating low HD5 is suggested to contribute to the age-related differences in the intestinal microbiota and may relate to increased risk of diseases in elderly people.

Decrease of α-defensin impairs intestinal metabolite homeostasis via dysbiosis in mouse chronic social defeat stress model.

Psychological stress has been reported to relate to dysbiosis, imbalance of the intestinal microbiota composition, and contribute to the onset and exacerbation of depression, though, underlying mechanisms of psychological stress-related dysbiosis have been unknown. It has been previously established that α-defensins, which are effector peptides of innate enteric immunity produced by Paneth cells in the small intestine, play an important role in regulation of the intestinal microbiota. However, the relationship between disruption of intestinal ecosystem and α-defensin under psychological stress is yet to be determined. Here we show using chronic social defeat stress (CSDS), a mouse depression model that (1) the exposure to CSDS significantly reduces α-defensin secretion by Paneth cells and (2) induces dysbiosis and significant composition changes in the intestinal metabolites. Furthermore, (3) they are recovered by administration of α-defensin. These results indicate that α-defensin plays an important role in maintaining homeostasis of the intestinal ecosystem under psychological stress, providing novel insights into the onset mechanism of stress-induced depression, and may further contribute to discovery of treatment targets for depression.

Simultaneous real-time analysis of Paneth cell and intestinal stem cell response to interferon-γ by a novel stem cell niche tracking method.

Paneth cells and Lgr5+ intestinal stem cells (Lgr5+ ISCs) constitute the stem cell niche and maintain small intestinal epithelial integrity by recognizing various niche factors derived from subepithelial cells and external antigens. Although it has been known that interferon-γ (IFN-γ), a Th1 cytokine, is associated with intestinal epithelial disruption during inflammation as a niche factor, dynamics of Paneth cells and Lgr5+ ISCs in response to IFN-γ remain to be understood. Here we show that CAG-tdTomato;Lgr5-EGFP (CT-LE) mice generated in this study enable to identify Paneth cells and Lgr5+ ISCs separately by fluorescence signals. Lgr5+ ISCs underwent cell death a little earlier than Paneth cells in response to IFN-γ by simultaneous tracking using CT-LE mice. In addition, the timing of cell death in most Paneth cells overlapped with Lgr5+ ISCs, suggesting that Paneth cell depletion is induced directly by IFN-γ. Taken together, we established a novel simultaneous stem cell niche tracking method and clarified the involvement of both Paneth cells and Lgr5+ ISCs in stem cell niche damage induced by IFN-γ, further contribute to understanding the mechanism for maintaining intestinal homeostasis by stem cell niche.

Expression and Localization of Paneth Cells and Their α-Defensins in the Small Intestine of Adult Mouse.

Paneth cells contribute to intestinal innate immunity by sensing bacteria and secreting α-defensin. In Institute of Cancer Research (ICR) mice, α-defensin termed cryptdin (Crp) in Paneth cells consists of six major isoforms, Crp1 to 6. Despite accumulating evidences that α-defensin functions in controlling the intestinal microbiota, topographical localization of Paneth cells in the small intestine in relation to functions of α-defensin remains to be determined. In this study, we examined the expression level of messenger RNA (mRNA) encoding six Crp-isoforms and Crp immunoreactivities using singly isolated crypts together with bactericidal activities of Paneth cell secretions from isolated crypts of duodenum, jejunum, and ileum. Here we showed that levels of Crp mRNAs in the single crypt ranged from 5 x 103 to 1 x 106 copies per 5 ng RNA. For each Crp isoform, the expression level in ileum was 4 to 50 times higher than that in duodenum and jejunum. Furthermore, immunohistochemical analysis of isolated crypts revealed that the average number of Paneth cell per crypt in the small intestine increased from proximal to distal, three to seven-fold, respectively. Both Crp1 and 4 expressed greater in ileal Paneth cells than those in duodenum or jejunum. Bactericidal activities in secretions of ileal Paneth cell exposed to bacteria were significantly higher than those of duodenum or jejunum. In germ-free mice, Crp expression in each site of the small intestine was attenuated and bactericidal activities released by ileal Paneth cells were decreased compared to those in conventional mice. Taken together, Paneth cells and their α-defensin in adult mouse appeared to be regulated topographically in innate immunity to control intestinal integrity.

Mycotoxin Deoxynivalenol Has Different Impacts on Intestinal Barrier and Stem Cells by Its Route of Exposure.

The different effects of deoxynivalenol (DON) on intestinal barrier and stem cells by its route of exposure remain less known. We explored the toxic effects of DON on intestinal barrier functions and stem cells after DON microinjection (luminal exposure) or addition to a culture medium (basolateral exposure) using three-dimensional mouse intestinal organoids (enteroids). The influx test using fluorescein-labeled dextran showed that basolateral DON exposure (1 micromolar (µM) disrupted intestinal barrier functions in enteroids compared with luminal DON exposure at the same concentration. Moreover, an immunofluorescence experiment of intestinal epithelial proteins, such as E-cadherin, claudin, zonula occludens-1 (ZO-1), and occludin, exhibited that only basolateral DON exposure broke down intestinal epithelial integrity. A time-lapse analysis using enteroids from leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescence protein (EGFP) transgenic mice and 5-ethynyl-2-deoxyuridine (EdU) assay indicated that only the basolateral DON exposure, but not luminal DON exposure, suppressed Lgr5+ stem cell count and proliferative cell ratio, respectively. These results revealed that basolateral DON exposure has larger impacts on intestinal barrier function and stem cells than luminal DON exposure. This is the first report that DON had different impacts on intestinal stem cells depending on the administration route. In addition, RNA sequencing analysis showed different expression of genes among enteroids after basolateral and luminal DON exposure.