Intestinal Stem Cell Activity Research Articles

Hypoxic preconditioning accelerates the healing of ischemic intestinal injury by activating HIF-1α/PPARα pathway-mediated fatty acid oxidation

Hypoxic preconditioning (HPC) has been shown to improve organ tolerance to subsequent severe hypoxia or ischemia. However, its impact on intestinal ischemic injury has not been well studied. In this study, we evaluated the effects of HPC on intestinal ischemia in rats. Intestinal rehabilitation, levels of fatty acid oxidation (FAO) by-products, intestinal stem cells (ISCs), levels of hypoxia-inducible factor 1 subunit α (HIF-1α) and its downstream genes such as peroxisome proliferator-activated receptor α (PPARα), and carnitine palmitoyltransferase 1a (CPT1A) were assessed at distinct time intervals following intestinal ischemia with or without the interference of HIF-1α. Our data showed that HPC facilitates the restoration of the intestinal structure and enhances the FAO, by boosting intestinal stem cells. Additionally, HIF-1α, PPARα, and CPT1A mRNA and their protein levels were generally up-regulated in the small intestine of HPC rats as compared to the control group. Our vitro experiment also shows low-oxygen induces highly levels of HIF-1α and its downstream genes, with a concurrent increase in FAO products in IEC-6 cells. Furthermore, the above phenomenon could be reversed by silencing HIF-1α. In conclusion, we hypothesize that HPC can stimulate the activation of intestinal stem cells via HIF-1α/PPARα pathway-mediated FAO, thereby accelerating the healing process post ischemic intestinal injury.

Btbd8 deficiency reduces susceptibility to colitis by enhancing intestinal barrier function and suppressing inflammation.

BTBD8 has been identified as a susceptible gene for inflammatory bowel diseases (IBD). However, the function of BTBD8 in normal development and IBD pathogenesis remains unknown. We administered drinking water with 3% dextran sodium sulfate (DSS) to wild-type (WT) and Btbd8 knockout (KO) mice for seven consecutive days to induce IBD. Subsequently, we further examined whether Btbd8 KO affects intestinal barrier and inflammation. We demonstrated that Btbd8 deficiency partially protects mice from DSS-induced IBD, even though no obvious phenotypes were observed in Btbd8 KO mice. Btbd8 deletion leads to strengthened tight junctions between intestinal epithelial cells, elevated intestinal stem cell activity, and enhanced mucus layer. All these three mechanisms work together to improve the intestinal barrier integrity in Btbd8 KO mice. In addition, Btbd8 deficiency mitigates inflammation by reducing the expression of IL-1β and IL-6 by macrophages. Our studies validate the crucial role of Btbd8 in IBD pathogenesis, and reveal that Btbd8 deficiency may ameliorate DSS-induced IBD through improving the intestinal barrier integrity, as well as suppressing inflammatory response mediated by macrophages. These findings suggest that Btbd8 could be a promising therapeutic target for the treatment of IBD.

The loss of antioxidant activities impairs intestinal epithelium homeostasis by altering lipid metabolism

Reactive oxygens species (ROS) are common byproducts of metabolic reactions and could be at the origin of many diseases of the elderly. Here we investigated the role of ROS in the renewal of the intestinal epithelium in mice lacking catalase (CAT) and/or nicotinamide nucleotide transhydrogenase (NNT) activities. Cat−/− mice have delayed intestinal epithelium renewal and were prone to develop necrotizing enterocolitis upon starvation. Interestingly, crypts lacking CAT showed fewer intestinal stem cells (ISC) and lower stem cell activity than wild-type. In contrast, crypts lacking NNT showed a similar number of ISCs as wild-type but increased stem cell activity, which was also impaired by the loss of CAT. No alteration in the number of Paneth cells (PCs) was observed in crypts of either Cat−/− or Nnt−/− mice, but they showed an evident decline in the amount of lysozyme. Cat deficiency caused fat accumulation in crypts, and a fall in the remarkable high amount of adipose triglyceride lipase (ATGL) in PCs. Notably, the low levels of ATGL in the intestine of Cat −/− mice increased after a treatment with the antioxidant N-acetyl-L-cysteine. Supporting a role of ATGL in the regulation of ISC activity, its inhibition halt intestinal organoid development. These data suggest that the reduction in the renewal capacity of intestine originates from fatty acid metabolic alterations caused by peroxisomal ROS.

Matrine reduced intestinal stem cell damage in eimeria necatrix-infected chicks via blocking hyperactivation of Wnt signaling

BackgroundCoccidiosis is a rapidly spreading and acute parasitic disease that seriously threatening the intestinal health of poultry. Matrine from leguminous plants has anthelmintic and anti-inflammatory properties. PurposeThis assay was conducted to explore the protective effects of Matrine and the AntiC (a Matrine compound) on Eimeria necatrix (EN)-infected chick small intestines and to provide a nutritional intervention strategy for EN injury. Study designThe in vivo (chick) experiment: A total of 392 one-day-old yellow-feathered broilers were randomly assigned to six groups in a 21-day study: control group, 350 mg/kg Matrine group, 500 mg/kg AntiC group, EN group, and EN + 350 mg/kg Matrine group, EN + 500 mg/kg AntiC group. The in vitro (chick intestinal organoids, IOs): The IOs were treated with PBS, Matrine, AntiC, 3 μM CHIR99021, EN (15,000 EN sporozoites), EN + Matrine, EN + AntiC, EN + Matrine + CHIR99021, EN + AntiC + CHIR99021. MethodsThe structural integrity of chicks jejunal crypt-villus axis was evaluated by hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM). And the activity of intestinal stem cells (ISCs) located in crypts was assessed by in vitro expansion advantages of a primary in IOs model. Then, the changes of Wnt/β-catenin signaling in jejunal tissues and IOs were detected by Real-Time qPCR,Western blotting and immunohistochemistry. ResultsThe results showed that dietary supplementation with Matrine or AntiC rescued the jejunal injury caused by EN, as indicated by increased villus height, reduced crypt hyperplasia, and enhanced expression of tight junction proteins. Moreover, there was less budding efficiency of the IOs expanded from jejunal crypts of chicks in the EN group than that in the Matrine and AntiC group, respectively. Further investigation showed that AntiC and Matrine inhibited EN-stimulated Wnt/β-catenin signaling. The fact that Wnt/β-catenin activation via CHIR99021 led to the failure of Matrine and AntiC to rescue damaged ISCs confirmed the dominance of this signaling. ConclusionOur results suggest that Matrine and AntiC inhibit ISC proliferation and promote ISC differentiation into absorptive cells by preventing the hyperactivation of Wnt/β-catenin signaling, thereby standardizing the function of ISC proliferation and differentiation, which provides new insights into mitigating EN injury by Matrine and AntiC.

Death receptor 5 is required for intestinal stem cell activity during intestinal epithelial renewal at homoeostasis

Intestinal epithelial renewal, which depends on the proliferation and differentiation of intestinal stem cells (ISCs), is essential for epithelial homoeostasis. Understanding the mechanism controlling ISC activity is important. We found that death receptor 5 (DR5) gene deletion (DR5-/-) mice had impaired epithelial absorption and barrier function, resulting in delayed weight gain, which might be related to the general reduction of differentiated epithelial cells. In DR5-/- mice, the expression of ISC marker genes, the number of Olfm4+ ISCs, and the number of Ki67+ and BrdU+ cells in crypt were reduced. Furthermore, DR5 deletion inhibited the expression of lineage differentiation genes driving ISC differentiation into enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Therefore, DR5 gene loss may inhibit the intestinal epithelial renewal by dampening ISC activity. The ability of crypts from DR5-/- mice to form organoids decreased, and selective DR5 activation by Bioymifi promoted organoid growth and the expression of ISC and intestinal epithelial cell marker genes. Silencing of endogenous DR5 ligand TRAIL in organoids down-regulated the expression of ISC and intestinal epithelial cell marker genes. So, DR5 expressed in intestinal crypts was involved in the regulation of ISC activity. DR5 deletion in vivo or activation in organoids inhibited or enhanced the activity of Wnt, Notch, and BMP signalling through regulating the production of Paneth cell-derived ISC niche factors. DR5 gene deletion caused apoptosis and DNA damage in transit amplifying cells by inhibiting ERK1/2 activity in intestinal crypts. Inhibition of ERK1/2 with PD0325901 dampened the ISC activity and epithelial regeneration. In organoids, when Bioymifi’s effect in activating ERK1/2 activity was completely blocked by PD0325901, its role in stimulating ISC activity and promoting epithelial regeneration was also eliminated. In summary, DR5 in intestinal crypts is essential for ISC activity during epithelial renewal under homoeostasis.

Host-Microbe Interactions Regulate Intestinal Stem Cells and Tissue Turnover in Drosophila.

With the activity of intestinal stem cells and continuous turnover, the gut epithelium is one of the most dynamic tissues in animals. Due to its simple yet conserved tissue structure and enteric cell composition as well as advanced genetic and histologic techniques, Drosophila serves as a valuable model system for investigating the regulation of intestinal stem cells. The Drosophila gut epithelium is in constant contact with indigenous microbiota and encounters externally introduced "non-self" substances, including foodborne pathogens. Therefore, in addition to its role in digestion and nutrient absorption, another essential function of the gut epithelium is to control the expansion of microbes while maintaining its structural integrity, necessitating a tissue turnover process involving intestinal stem cell activity. As a result, the microbiome and pathogens serve as important factors in regulating intestinal tissue turnover. In this manuscript, I discuss crucial discoveries revealing the interaction between gut microbes and the host's innate immune system, closely associated with the regulation of intestinal stem cell proliferation and differentiation, ultimately contributing to epithelial homeostasis.

Krüppel-like factor 5 activates chick intestinal stem cell and promotes mucosal repair after impairment

ABSTRACTThe mucosal renewal, which depends on the intestinal stem cell (ISC) activity, is the foundation of mucosal repairment. Importantly, activation of reserve ISCs (rISCs) plays a vital role in initiating mucosal repair after injury. However, the underlying regulatory mechanism of rISCs activation in chickens remains unclear. In this study, immediately after lipopolysaccharide (LPS) challenge, mitochondrial morphological destruction and dysfunction appeared in the crypt, accompanied by decreased epithelial secretion (decreased Muc2 mRNA abundance and LYSOZYME protein level). However, immediately after mucosal injury, the mucosal renewal accelerated, as indicated by the increased BrdU positive rate, proliferating cell nuclear antigen (PCNA) protein level and mRNA abundance of cell cycle markers (Ccnd1, Cdk2). Concerning the ISCs activity, during the early period of injury, there appeared a reduction of active ISCs (aISCs) marker Lgr5 mRNA and protein, and an increasing of rISCs marker Hopx mRNA and protein. Strikingly, upon LPS challenge, increased mRNA transcriptional level of Krüppel-like factor 5 (Klf5) was detected in the crypt. Moreover, under LPS treatment in organoids, the KLF5 inhibitor (ML264) would decrease the mRNA and protein levels of Stat5a and Hopx, the STAT5A inhibitor (AC-4-130) would suppress the Lgr5 mRNA and protein levels. Furthermore, the Dual-Luciferase Reporter assay confirmed that, KLF5 would bind to Hopx promoter and activate the rISCs, STAT5A would trigger Lgr5 promoter and activate the aISCs. Collectively, KLF5 was upregulated during the early period of injury, further activate the rISCs directly and activate aISCs via STAT5A indirectly, thus initiate mucosal repair after injury.

Control of Paneth cell function by HuR regulates gut mucosal growth by altering stem cell activity.

Rapid self-renewal of the intestinal epithelium requires the activity of intestinal stem cells (ISCs) that are intermingled with Paneth cells (PCs) at the crypt base. PCs provide multiple secreted and surface-bound niche signals and play an important role in the regulation of ISC proliferation. Here, we show that control of PC function by RNA-binding protein HuR via mitochondria affects intestinal mucosal growth by altering ISC activity. Targeted deletion of HuR in mice disrupted PC gene expression profiles, reduced PC-derived niche factors, and impaired ISC function, leading to inhibited renewal of the intestinal epithelium. Human intestinal mucosa from patients with critical surgical disorders exhibited decreased levels of tissue HuR and PC/ISC niche dysfunction, along with disrupted mucosal growth. HuR deletion led to mitochondrial impairment by decreasing the levels of several mitochondrial-associated proteins including prohibitin 1 (PHB1) in the intestinal epithelium, whereas HuR enhanced PHB1 expression by preventing microRNA-195 binding to the Phb1 mRNA. These results indicate that HuR is essential for maintaining the integrity of the PC/ISC niche and highlight a novel role for a defective PC/ISC niche in the pathogenesis of intestinal mucosa atrophy.

Paneth cells protect intestinal stem cell niche to alleviate deoxynivalenol-induced intestinal injury

Deoxynivalenol (DON) is a common toxin in grains and feeds, and DON exposure triggers severe small intestinal injury and inflammation, which harms the health of humans and livestock. DON treatment leads to a decrease in Paneth cells, whereas the role of Paneth cells in DON-induced intestinal injury is poorly understood. We utilized dithizone (40 mg/kg) to keep murine Paneth cell number at a low level. The results showed that dithizone-mediated long-term disruption of Paneth cells aggravated intestinal injury, intestinal stem cell (ISC) loss, and microbiota disorder in DON (2 mg/kg)-treated mice. Unexpectedly, the number of goblet cells and proliferative cells was boosted in mice treated with dithizone and DON. After dithizone and DON treatments, the Firmicutes/Bacteroidetes (F/B) ratio was reduced, and the increased abundance of Dubosiella and the decreased abundance of Lactobacillus were observed in mice. The functional recovery of Paneth cells by lysozyme (200 U/day) supplementation improved intestinal injury and ISC loss in mice after DON challenge. In addition, lysozyme also promoted the growth and ISC activity of intestinal organoids. Taken together, these results demonstrate the protective role of Paneth cells in DON-induced intestinal injury. Our study raises a novel target, Paneth cell, for the treatment of DON exposure.

Glutamine boosts intestinal stem cell-mediated small intestinal epithelial development during early weaning: Involvement of WNT signaling.

Early weaning usually causes small intestine epithelial development abnormality, increasing the risk of gastrointestinal diseases. Glutamine (Gln), enriching in plasma and milk, is widely reported to benefit intestinal health. However, whether Gln affects intestinal stem cell (ISC) activity in response to early weaning is unclear. Here, both the early weaning mice and intestinal organoids were used to study the role of Gln in regulating ISC activities. Results showed that Gln ameliorated early weaning-induced epithelial atrophy and augmented the ISC-mediated epithelial regeneration. Gln deprivation disabled ISC-mediated epithelial regeneration and crypt fission invitro. Mechanistically, Gln augmented WNT signaling in a dose-dependent manner to regulate ISC activity, while WNT signaling blockage abolished the effects of Gln on ISCs. Together, Gln accelerates stem cell-mediated intestinal epithelial development associated with the augmentation of WNT signaling, which provides novel insights into the mechanism by which Gln promotes intestinal health.

Regulation of intestinal stem cell activity by a mitotic cell cycle regulator Polo in Drosophila.

Maintaining a definite and stable pool of dividing stem cells plays an important role in organ development. This process requires an appropriate progression of mitosis for proper spindle orientation and polarity to ensure the ability of stem cells to proliferate and differentiate correctly. Polo-like kinases (Plks)/Polo are the highly conserved serine/threonine kinases involved in the initiation of mitosis as well as in the progression of the cell cycle. Although numerous studies have investigated the mitotic defects upon loss of Plks/Polo in cells, little is known about the in vivo consequences of stem cells with abnormal Polo activity in the context of tissue and organism development. The current study aimed to investigate this question using the Drosophila intestine, an organ dynamically maintained by the intestinal stem cells (ISCs). The results indicated that the polo depletion caused a reduction in the gut size due to a gradual decrease in the number of functional ISCs. Interestingly, the polo-deficient ISCs showed an extended G2/M phase and aneuploidy and were subsequently eliminated by premature differentiation into enterocytes (ECs). In contrast, the constitutively active Polo (poloT182D) suppressed ISC proliferation, induced abnormal accumulation of β-tubulin in cells, and drove ISC loss via apoptosis. Therefore, Polo activity should be properly maintained for optimal stem cell function. Further analysis suggested that polo was a direct target gene of Sox21a, a Sox transcription factor that critically regulates stem cell activity. Together, this study provided a novel perspective on the correlation between the progression of mitosis and the ISC function in Drosophila.

Inulin diet uncovers complex diet-microbiota-immune cell interactions remodeling the gut epithelium

BackgroundThe continuous proliferation of intestinal stem cells followed by their tightly regulated differentiation to epithelial cells is essential for the maintenance of the gut epithelial barrier and its functions. How these processes are tuned by diet and gut microbiome is an important, but poorly understood question. Dietary soluble fibers, such as inulin, are known for their ability to impact the gut bacterial community and gut epithelium, and their consumption has been usually associated with health improvement in mice and humans. In this study, we tested the hypothesis that inulin consumption modifies the composition of colonic bacteria and this impacts intestinal stem cells functions, thus affecting the epithelial structure.MethodsMice were fed with a diet containing 5% of the insoluble fiber cellulose or the same diet enriched with an additional 10% of inulin. Using a combination of histochemistry, host cell transcriptomics, 16S microbiome analysis, germ-free, gnotobiotic, and genetically modified mouse models, we analyzed the impact of inulin intake on the colonic epithelium, intestinal bacteria, and the local immune compartment.ResultsWe show that the consumption of inulin diet alters the colon epithelium by increasing the proliferation of intestinal stem cells, leading to deeper crypts and longer colons. This effect was dependent on the inulin-altered gut microbiota, as no modulations were observed in animals deprived of microbiota, nor in mice fed cellulose-enriched diets. We also describe the pivotal role of γδ T lymphocytes and IL-22 in this microenvironment, as the inulin diet failed to induce epithelium remodeling in mice lacking this T cell population or cytokine, highlighting their importance in the diet-microbiota-epithelium-immune system crosstalk.ConclusionThis study indicates that the intake of inulin affects the activity of intestinal stem cells and drives a homeostatic remodeling of the colon epithelium, an effect that requires the gut microbiota, γδ T cells, and the presence of IL-22. Our study indicates complex cross kingdom and cross cell type interactions involved in the adaptation of the colon epithelium to the luminal environment in steady state.-dZ131ovLvfeoRbZ-Pz4qeVideo

Dietary supplementation with probiotics increases growth performance, improves the intestinal mucosal barrier and activates the Wnt/β-catenin pathway activity in chicks.

Probiotics comprise effective feed additives that can replace antibiotics in animal livestock production. However, mono-strain probiotics appear less effective because of their instability. Therefore, the present study aimed to investigate dietary supplementation with compound probiotics (CPP) on growth performance, diarrhea rate and intestinal mucosal barrier, as well as the possible molecular mechanism, in chicks. In total, 360 1-day-old chicks of the Hy-Line Brown Chicks were randomly divided into the control group (CON, basal diet), chlortetracycline group (500 mg kg-1 CTC) and compound probiotics group (1000 mg kg-1 CPP, consisting of Bacillus subtilis, Bacillus licheniformis, Enterococcus faecium and yeast). The experiment period was 56 days. The results showed that, in comparison with the CON group, CPP significantly increased the average daily feed intake and average daily gain of chicks and reduced diarrhea (P < 0.05). The probiotic group exhibited increased immune organ (i.e. spleen and thymus) mass and increased levels of serum immunoglobulin (Ig)A, IgM and IgG (P < 0.05) compared to the CTC group. In addition, the jejunal mass and morphology were improved in the probiotic group (P < 0.05). Moreover, CPP reinforced jejunal barrier function, as indicated by increased transepithelial electrical resistance, protein expression of occludin and claudin-1, and diamine oxidase levels in the jejunum (P < 0.05). Likewise, enhanced fluorescence signals of proliferating cell nuclear antigen-labeled mitotic cells and villin-labeled absorptive cells in the jejunum (P < 0.05) suggested that CPP promoted intestinal stem cells activity. Mechanistically, the Wnt/β-catenin signaling pathway, including β-catenin, TCF4, c-Myc, cyclin D1 and Lgr5, was amplified in the jejunum by CPP addition (P < 0.05). The present study demonstrated that dietary supplementation with CPP reinforced the jejunal epithelial integrity by activating Wnt/β-catenin signaling and enhanced immune function in chicks. © 2023 Society of Chemical Industry.

Early weaning causes small intestinal atrophy by inhibiting the activity of intestinal stem cells: involvement of Wnt/β-catenin signaling

BackgroundEarly weaning and shorter breastfeeding duration are applied by a proportion of young mothers, especially in the social spheres of poverty-stricken areas. Early childhood is a critical period for intestinal development, which is driven by intestinal stem cells (ISCs). However, how early weaning practice affects the function of ISCs to mediate intestinal development remains unclear.MethodsWe established an excellent early weaning mice model that has significant intestinal atrophy and growth arrest symptoms to explore the responses of ISCs to early weaning. The primary and passaged intestinal organoids from the suckling or early weaning mice were cultured to explore the underlying mechanism of early weaning affecting the ISCs.ResultsEarly weaning depressed the self-renewal of ISCs and attenuated the activity of ISCs-driven intestinal epithelial regeneration and crypt expansion in vivo and ex-vivo. Further results showed that early weaning retarded the differentiation of ISCs into transit-amplifying cells and Paneth cells, and accelerated the apoptosis of villous epithelial cells, jointly leading to intestinal epithelial atrophy. Mechanistically, early weaning inhibited Wnt signaling in ISCs, while an exogenous Wnt amplifier restored ISCs’ function in ex-vivo.ConclusionOur findings indicate that early weaning depresses the activity of ISCs via attenuating Wnt/β-catenin signaling and triggers the proinflammatory cytokines TNF-α, IL-1β, IL-6, and IL-17 in jejunum, thereby impeding ISCs-driven epithelial regeneration and intestinal growth, which may provide a basal theory for the development of infant nutrients targeting stem cells to alleviate early weaning-induced intestinal problems.

Quercetin Mitigates Radiation-Induced Intestinal Injury and Promotes Intestinal Regeneration via Nrf2-Mediated Antioxidant Pathway1.

Radiation-induced intestinal injury is one the most common adverse events of radiotherapy, which can severely affect quality of life. There are currently no effective preventive and therapeutic options for this disorder. Quercetin is a natural flavonoid found in common food species, with the characteristics of antioxidative, anti-inflammatory, and anti-cancerous activity. However, the role of quercetin on radiation-induced intestinal injury and the underlying mechanism remains poorly understood. In this study, we found quercetin treatment can improve the survival rate of mice after a single-dose (10 Gy) abdominal irradiation. Quercetin-pretreated mice significantly reduced radiation-induced DNA damage and intestinal epithelium cell apoptosis. In addition, quercetin also improved the proliferation activity of intestinal stem cells and promoted intestine epithelium repair after irradiation. Further studies demonstrated that quercetin treatment curtailed radiation-induced reactive oxygen species generation via regulating Nrf2 signaling in intestinal epithelium cells. Furthermore, treatment with Nrf2 inhibitor, could reverse the above effects. Altogether, quercetin can ameliorate radiation-induced intestine injury via regulating Nrf2 signaling, scavenging free radicals, and promoting intestinal epithelium repair.

Enteroendocrine Cells Protect the Stem Cell Niche by Regulating Crypt Metabolism in Response to Nutrients

The intestinal stem cell niche is exquisitely sensitive to changes in diet, with high-fat diet, caloric restriction, and fasting resulting in altered crypt metabolism and intestinal stem cell function. Unlike cells on the villus, cells in the crypt are not immediately exposed to the dynamically changing contents of the lumen. We hypothesized that enteroendocrine cells (EECs), which sense environmental cues and in response release hormones and metabolites, are essential for relaying the luminal and nutritional status of the animal to cells deep in the crypt. We used the tamoxifen-inducible VillinCreERT2 mouse model to deplete EECs (Neurog3fl/fl) from adult intestinal epithelium and we generated human intestinal organoids from wild-type and NEUROGENIN 3 (NEUROG3)-null human pluripotent stem cells. We used indirect calorimetry, 1H-Nuclear Magnetic Resonance (NMR) metabolomics, mitochondrial live imaging, and the Seahorse bioanalyzer (Agilent Technologies) to assess metabolism. Intestinal stem cell activity was measured by proliferation and enteroid-forming capacity. Transcriptional changes were assessed using 10x Genomics single-cell sequencing. Loss of EECs resulted in increased energy expenditure in mice, an abundance of active mitochondria, and a shift of crypt metabolism to fatty acid oxidation. Crypts from mouse and human intestinal organoids lacking EECs displayed increased intestinal stem cell activity and failed to activate phosphorylation of downstream target S6 kinase ribosomal protein, a marker for activity of the master metabolic regulator mammalian target of rapamycin (mTOR). These phenotypes were similar to those observed when control mice were deprived of nutrients. EECs are essential regulators of crypt metabolism. Depletion of EECs recapitulated a fasting metabolic phenotype despite normal levels of ingested nutrients. These data suggest that EECs are required to relay nutritional information to the stem cell niche and are essential regulators ofintestinal metabolism.

Abstract B023: Sox9 drives an aberrant transcriptional program impeding intestinal differentiation in colorectal cancer initiation

Abstract Despite the implementation of screening and preventative strategies, colorectal cancer (CRC) remains the second most deadly cancer worldwide and is responsible for an alarming trend of increasing prevalence among younger patients. Inappropriate WNT activation is the initiating step in sporadic CRC development, typically through deleterious mutations in the pathway negative regulator Adenomatous polyposis coli (APC). WNT pathway hinders whereas transforming growth factor (TGF-β)/bone morphogenetic protein (BMP) signaling supports differentiation of progenitors into mature enterocytes, establishing a crypt-villus gradient. Genomic alterations that dysregulate intestinal stem cell differentiation are central to CRC development. Impairment of differentiation and inappropriate Sox9 overexpression are conserved events in CRC initiation based on the evaluation of two genetically engineered mouse models, two carcinogen-induced mouse models, and a patient with familial adenomatous polyposis (FAP), a hereditary polyposis syndrome in which a mutant copy of APC is inherited. Single cell RNA-sequencing (scRNA-seq) of adenomas from an ApcKO mouse model and FAP sample implicates an aberrant stem cell-like program (herein referred to as AbSC) as a key aspect of CRC initiation. The AbSc program is characterized by selective intestinal stem cell activity, indiscriminate attenuation of differentiated lineages, and aberrant activation of genes associated with fetal intestinal development. Histopathology and scRNA-seq of adenomas and derivative organoids from a patient with FAP demonstrated a partial block in differentiation and confirmed reactivation of genes reserved for intestinal development. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) of APCKO epithelial cells revealed increased chromatin accessibility at fetal intestinal genes. Genetic inactivation of Sox9 prevented adenoma formation in ApcKO mice and induced differentiation of ApcKO organoids, obstructing the emergence of the ApcKO transcriptional state, including reactivation of a fetal-like intestinal program, restored multi-lineage differentiation, and markedly reduced the gained chromatin accessibility at developmental genes in neoplasia. These studies indicate that an aberrant transcriptional state hindering intestinal differentiation mediated by Sox9 is an early conserved event in CRC and carry important implications for developing therapeutics directed at inducing intestinal differentiation. Citation Format: Pratyusha Bala, Jonathan P. Rennhack, Clare Morris, Daulet Aitymbayev, Sydney M. Moyer, Gina Nicole Duronio, Paul Doan, William C. Hahn, Nilay S. Sethi. Sox9 drives an aberrant transcriptional program impeding intestinal differentiation in colorectal cancer initiation [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B023.

Lactobacillus salivarius Promotion of Intestinal Stem Cell Activity in Hens Is Associated with Succinate-Induced Mitochondrial Energy Metabolism.

Currently, the regulation of Lactobacillus on intestinal stem cells (ISCs) attracts broad attention, but their active ingredients and the underlying mechanism are worthy of further study. Previously, host intestinal commensal bacteria were verified to drive the differentiation of ISCs. In this study, the strong bacteriostatic activity of Lactobacillus salivarius and Lactobacillus agilis were illustrated, and the components (supernatant, precipitation) of L. salivarius or L. agilis were further demonstrated to decrease the differentiation of ISCs in vivo. Interestingly, antibiotics feeding decreased ISCs differentiation in vivo as well. However, the administration of L. salivarius supernatant following antibiotics feeding was shown to promote ISCs differentiation dramatically when compared with the antibiotics feeding group, indicating that some active ingredients existed in its supernatant to promote ISCs activity. Strikingly, in vitro, the treatment of L. salivarius supernatant was further confirmed to promote the intestinal organoids' size, budding, and LGR5 expression. Next, the metabolomics analysis of Lactobacilli' supernatants suggested that succinate might be a crucial metabolite to promote ISCs activity. Further, the succinate treatment in vitro (1000 μM) and in vivo (50 mM) was confirmed to enhance the expression of LGR5 and PCNA. SLC13A3 (a sodium/dicarboxylate cotransporter) was detected in the intestinal organoids and demonstrated to transport succinate into ISCs, as confirmed by the contact of FITC-succinate with ISCs nucleus. Subsequently, high mitochondrial membrane potential and reactive oxygen species levels appeared in the intestinal organoids upon succinate treatment. Collectively, the promotion of L. salivarius on ISCs activity is associated with succinate-induced mitochondrial energy metabolism. IMPORTANCE In our previous study, Lactobacillus salivarius and Lactobacillus agilis were demonstrated to regulate intestinal stem cell activity in hens, but their active ingredients and the underlying mechanism remain unclear. In this study, L. salivarius supernatant was shown to directly promote intestinal stem cell activity. Furthermore, the succinate (a critical metabolite of L. salivarius) was screened out to promote intestinal stem cell activity. Moreover, the succinate was confirmed to enter intestinal stem cells and induce high mitochondrial energy metabolism, finally promoting intestinal stem cell activity. These findings will advance uncovering the mechanism by which Lactobacillus regulate intestinal stem cell activity in chickens.

PSIX-20 Nrf2 Pathway Mediates Resistance to Weaning Stress-Induced Intestinal Injury in Piglets of Different Breeds

Abstract The intestine is the central organ of early weaning stress in piglets; however, the response of intestinal stem cells (ISCs) that drive epithelial renewal between the different breeds of piglets is not yet known. Therefore, this investigation was to explore the sensitivity of ISCs to early weaning stress. Taoyuan Black and Duroc piglets were slaughtered at 21 days of age (on the day of weaning) and 24 days of age (3 days after weaning) with 10 pigs, respectively. And the results showed that Taoyuan Black pigs are more tolerant to early weaning stress, while the mucosal mass of jejunum was significantly decreased in 24-day-old Duroc piglets compared with the 21-day-old Duroc piglets (P &amp;lt; 0.05). The weaning stress damaged the jejunal structure, decreased the expression of tight junction related proteins ZO-1, Occludin, and Claudin-1, and increased intestinal permeability in Duroc piglets (P &amp;lt; 0.05). The ex vivo intestinal organoids (IOs) derived from the jejunal crypt of Duroc piglets after weaning exhibited lower expansion efficiency (P &amp;lt; 0.05). Furthermore, the levels of proliferating cell marker PCNA, terminally differentiated cell marker KRT20, absorptive cell marker Villin, goblet cell marker MUC2, and Paneth cell marker lysozyme were significantly reduced in the jejunum, crypt, and jejunal organoids of 24-day-old Duroc (P &amp;lt; 0.05). ROS levels in the jejunum of Duroc piglets were increased along with the inhibition of the Nrf2 pathway compared with 21-day-old Duroc piglets (P &amp;lt; 0.05). Collectively, the Taoyuan Black pigs had stronger tolerance to weaning stress; however early weaning suppressed the Nrf2 pathway, decreased proliferation and differentiation activity of ISCs, and disrupted intestinal homeostasis in Duroc piglets.

L-glutamate requires β-catenin signalling through Frizzled7 to stimulate porcine intestinal stem cell expansion.

Intestinal stem cells (ISCs) decode and coordinate various types of nutritional information from the diet to support the crypt-villus axis architecture, but how specific dietary molecules affect intestinal epithelial homeostasis remains unclear. In the current study, L-glutamate (Glu) supplementation in either a nitrogen-free diet (NFD) or a corn-soybean meal diet (CSMD) stimulated gut growth and ISC expansion in weaned piglets. Quantitative proteomics screening identified the canonical Wnt signalling pathway as a central regulator of intestinal epithelial development and ISC activity in vivo. Importantly, the Wnt transmembrane receptor Frizzled7 (FZD7) was upregulated in response to dietary Glu patterns, and its perturbations in intestinal organoids (IOs) treated with a specific inhibitor and in FZD7-KO IPEC-J2 cells disrupted the link between Glu inputs and β-catenin signalling and a subsequent reduction in cell viability. Furthermore, co-localization, coimmunoprecipitation (Co-IP), isothermal titration calorimetry (ITC), and microscale thermophoresis (MST) revealed that Glu served as a signalling molecule directly bound to FZD7. We propose that FZD7-mediated integration of the extracellular Glu signal controls ISC proliferation and differentiation, which provides new insights into the crosstalk of nutrients and ISCs.