Small Intestinal Crypts Research Articles

Lactobacillus rhamnosus GG increases Toll-like receptor 3 gene expression in murine small intestine ex vivo and in vivo.

Administration of Lactobacillus rhamnosus GG (LGG) has been reported to be therapeutically effective against acute secretory diarrhoea resulting from the structural and functional intestinal mucosal lesions induced by rotavirus infection; however, the underlying mechanisms remain to be completely elucidated. Because Toll-like receptor 3 (TLR3) plays a key role in the innate immune responses following the recognition of rotavirus, the present study examined whether LGG influences TLR3 gene expression in murine small intestine ex vivo and in vivo. We employed cultured intestinal organoids derived from small intestinal crypts as an ex vivo tissue model. LGG supplementation increased TLR3 mRNA levels in the intestinal organoids, as estimated by quantitative real-time polymerase chain reaction. Likewise, single and 7-day consecutive daily administrations of LGG increased TLR3 mRNA levels in the small intestine of C57BL/6N mice. The mRNA levels of other TLRs were not substantially altered both ex vivo and in vivo. In addition, LGG supplementation increased the mRNA levels of an antiviral type 1 interferon, interferon-α (IFN-α), and a neutrophil chemokine, CXCL1, upon stimulation with a synthetic TLR3 ligand, poly(I:C) in the intestinal organoids. LGG administration did not alter IFN-α and CXCL1 mRNA levels in the small intestine in vivo. Supplementation of other bacterial strains, Bifidobacterium bifidum and Lactobacillus paracasei, failed to increase TLR3 and poly(I:C)-stimulated CXCL1 mRNA levels ex vivo. We propose that upregulation of TLR3 gene expression may play a pivotal role in the therapeutic efficacy of LGG against rotavirus-associated diarrhoea. In addition, we demonstrated that intestinal organoids may be a promising ex vivo tissue model for investigating host-pathogen interactions and the antiviral action of probiotics in the intestinal epithelium.

The synergystic effects of omega-3 fatty acids against 5-fluorouracil-induced mucosal impairment in mice

Anti-cancer pharmaceuticals frequently have adverse side effects on patients such as gastrointestinal involvement limiting their clinical applications. These effects may be controlled by nutritional interventions, however, there are few studies that have shown any mechanistic effects. In this study, we examined effects of diet enhanced with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on 5-fluorouracil (5-FU)-induced intestinal impairment and immunity in mice. C57Bl6 mice were randomized to control diet, control diet + EPA, control + DHA, control + fish oil, or diet enchanced with DHA/EPA. After seven days of each respective diet, mice, excluding those in the sham group, were treated with 10 mg/kg/day 5-FU for 7 days. The effects of 5-FU-induced impairment in the small intestine were assessed using cytokine concentrations in serum and tissue, secretory immunoglobulin (Ig) A, diamine oxidase (DAO) activity, the length of the small intestine, and the expression of apoptosis signaling genes. The EPA/DHA-enhanced diet resulted in the most beneficial, synergystic and protective effect against 5-FU induced weight loss. Protection against inflammation, impaired intestinal function, and immunity of the small intestine were also observed. Individually, a DHA-enriched diet demonstrated a protective effect against 5-FU damage with longer small intestine mucosal and crypt lengths, greater DAO activity, and higher IgA concentrations, whereas the EPA-enriched diet resulted in decreased inflammatory cytokine concentrations in both plasma and small intestine and expression of apoptosis target genes. In conclusion, a diet enhanced with EPA and DHA results in synergism protecting against the detrimental effects of 5-FU and limiting chemotherapy induced mucosal impairment.

BVES Regulates Intestinal Stem Cell Programs and Intestinal Crypt Viability after Radiation.

Blood vessel epicardial substance (BVES/Popdc1) is a junctional-associated transmembrane protein that is underexpressed in a number of malignancies and regulates epithelial-to-mesenchymal transition. We previously identified a role for BVES in regulation of the Wnt pathway, a modulator of intestinal stem cell programs, but its role in small intestinal (SI) biology remains unexplored. We hypothesized that BVES influences intestinal stem cell programs and is critical to SI homeostasis after radiation injury. At baseline, Bves(-/-) mice demonstrated increased crypt height, as well as elevated proliferation and expression of the stem cell marker Lgr5 compared to wild-type (WT) mice. Intercross with Lgr5-EGFP reporter mice confirmed expansion of the stem cell compartment in Bves(-/-) mice. To examine stem cell function after BVES deletion, we used ex vivo 3D-enteroid cultures. Bves(-/-) enteroids demonstrated increased stemness compared to WT, when examining parameters such as plating efficiency, stem spheroid formation, and retention of peripheral cystic structures. Furthermore, we observed increased proliferation, expression of crypt-base columnar "CBC" and "+4" stem cell markers, amplified Wnt signaling, and responsiveness to Wnt activation in the Bves(-/-) enteroids. Bves expression was downregulated after radiation in WT mice. Moreover, after radiation, Bves(-/-) mice demonstrated significantly greater SI crypt viability, proliferation, and amplified Wnt signaling in comparison to WT mice. Bves(-/-) mice also demonstrated elevations in Lgr5 and Ascl2 expression, and putative damage-responsive stem cell populations marked by Bmi1 and TERT. Therefore, BVES is a key regulator of intestinal stem cell programs and mucosal homeostasis. Stem Cells 2016;34:1626-1636.

Brachyury identifies a class of enteroendocrine cells in normal human intestinal crypts and colorectal cancer.

Normal homeostasis of adult intestinal epithelium and repair following tissue damage is maintained by a balance of stem and differentiated cells, many of which are still only poorly characterised. Enteroendocrine cells of the gut are a small population of differentiated, secretory cells that are critical for integrating nutrient sensing with metabolic responses, dispersed amongst other epithelial cells. Recent evidence suggests that sub-sets of secretory enteroendocrine cells can act as reserve stem cells. Given the link between cells with stem-like properties and cancer, it is important that we identify factors that might provide a bridge between the two. Here, we identify a sub-set of chromogranin A-positive enteroendocrine cells that are positive for the developmental and cancer-associated transcription factor Brachyury in normal human small intestinal and colonic crypts. Whilst chromogranin A-positive enteroendocrine cells are also Brachyury-positive in colorectal tumours, expression of Brachyury becomes more diffuse in these samples, suggesting a more widespread function in cancer. The finding of the developmental transcription factor Brachyury in normal adult human intestinal crypts may extend the functional complexity of enteroendocrine cells and serves as a platform for assessment of the molecular processes of intestinal homeostasis that underpins our understanding of human health, cancer and aging.

Protective effect of vitamin E against ethanol-induced small intestine damage in rats.

The role of oxidative stress and inflammatory reaction has been reported in various ethanol-induced complications. The purpose of this study was to evaluate the effect of ethanol-induced structural alteration, oxidative stress, and inflammatory reaction on the small intestine of rats, and plausible protective effect of vitamin E to determine whether it inhibits the abnormality induced by ethanol in the small intestine. Twenty-four male wistar rats were divided into three groups, namely: Control, ethanol, and vitamin E treated ethanol groups. After six weeks of treatment, the small intestine length, villus height, crypt depth and muscular layer thickness, oxidative stress, and inflammatory parameters showed significant changes in the ethanol treated group compared to the control group. Vitamin E consumption along with ethanol ameliorated structural alteration of the small intestine and reduced the elevated amount of oxidative stress and inflammatory markers such as protein carbonyl, OX-LDL, IL-6, Hcy, and TNF-α. Furthermore, their total antioxidant capacity was increased significantly compared to that of the ethanol group. These findings indicate that ethanol induces the small intestine abnormality by oxidative and inflammatory stress, and that these effects can be alleviated by using vitamin E as an antioxidant and anti-inflammatory molecule.

Analysis of Cell Death Induction in Intestinal Organoids In Vitro.

The intestinal epithelium has an important function in the absorption of nutrients contained in the food. Furthermore, it also has an important barrier function, preventing luminal pathogens from entering the bloodstream. This single cell layer epithelium is quite sensitive to various cell death-promoting triggers, including drugs, irradiation, and TNF family members, leading to loss of barrier integrity, epithelial erosion, inflammation, malabsorption, and diarrhea. In order to assess the intestinal epithelium-damaging potential of treatments and substances specific test systems are required. As intestinal tumor cell lines are a poor substitute for primary intestinal epithelial cells, and in vivo experiments in mice are costly and often unethical, the use of intestinal organoids cultured from intestinal crypts provide an ideal tool to study cell death induction and mechanisms in primary intestinal epithelial cells. This protocol describes the isolation and culture of intestinal organoids from murine small intestinal crypts, and the quantitative assessment of cell death induction in these organoids.

Theoretical cross‐comparative analysis on dynamics of small intestine and colon crypts during cancer initiation

Epigenetics is emerging as a fundamentally important area of biological and medical research that has implications for our understanding of human diseases including cancer, autoimmune and neuropsychiatric disorders. In the context of recent efforts on personalised medicine, a novel research direction is concerned with identification of intra-individual epigenetic variation linked to disease predisposition and development, i.e. epigenome-wide association studies. A computational model has been developed to describe the dynamics and structure of human intestinal crypts and to perform a comparative analysis on aberrant DNA methylation level induced in these during cancer initiation. The crypt framework, AgentCrypt, is an agent-based model of crypt dynamics, which handles intra- and inter-dependencies. In addition, the AgentCrypt model is used to investigate the effect of a set of potential inhibitors with respect to methylation modification in intestinal tissue during initiation of disease. Methylation level decrease over a relatively short period of 90 days is marked for the colon compared to the small intestine, although similar alterations are induced in both tissues. In addition, inhibitor effect is notable for abnormal crypt groups, with largest average methylation differences observed ≈0.75% lower in the colon and ≈0.79% lower in the small intestine with inhibitor present.

MTOR disruption causes intestinal epithelial cell defects and intestinal atrophy postinjury in mice.

Intestinal stem cells (ISCs) drive small intestinal epithelial homeostasis and regeneration. Mechanistic target of rapamycin (mTOR) regulates stem and progenitor cell metabolism and is frequently dysregulated in human disease, but its physiologic functions in the mammalian small intestinal epithelium remain poorly defined. We disrupted the genes mTOR, Rptor, Rictor, or both Rptor and Rictor in mouse ISCs, progenitors, and differentiated intestinal epithelial cells (IECs) using Villin-Cre. Mutant tissues and wild-type or heterozygous littermate controls were analyzed by histologic immunostaining, immunoblots, and proliferation assays. A total of 10 Gy irradiation was used to injure the intestinal epithelium and induce subsequent crypt regeneration. We report that mTOR supports absorptive enterocytes and secretory Paneth and goblet cell function while negatively regulating chromogranin A-positive enteroendocrine cell number. Through additional Rptor, Rictor, and Rptor/Rictor mutant mouse models, we identify mechanistic target of rapamycin complex 1 as the major IEC regulatory pathway, but mechanistic target of rapamycin complex 2 also contributes to ileal villus maintenance and goblet cell size. Homeostatic adult small intestinal crypt cell proliferation, survival, and canonical wingless-int (WNT) activity are not mTOR dependent, but Olfm4(+) ISC/progenitor population maintenance and crypt regeneration postinjury require mTOR. Overall, we conclude that mTOR regulates multiple IEC lineages and promotes stem and progenitor cell activity during intestinal epithelium repair postinjury.

Porcine deltacoronavirus induces apoptosis in swine testicular and LLC porcine kidney cell lines in vitro but not in infected intestinal enterocytes in vivo

We compared the mechanisms of porcine delatacoronavirus (PDCoV) induced death of infected enterocytes in vivo and infected LLC porcine kidney (LLC-PK) and swine testicular (ST) cells in vitro. We conducted histologic analysis and immunofluorescence (IF) staining for the detection of PDCoV antigens, and TUNEL assay in singly or serially cut tissue sections from the small and large intestines of four, 11- to 14-day-old gnotobiotic pigs, inoculated orally with 8.8–11.0log10 genomic equivalents (GE) of US PDCoV strains OH-FD22 or OH-FD100 (n=3), or mock (n=1). Similar comparative assays were done on LLC-PK and ST cells inoculated with the cell-adapted PDCoV strain OH-FD22-P44 (passage 44) in cell culture medium with 2.5–10μg/ml of trypsin and 1% pancreatin, respectively. At post-inoculation days 3–4, infected pigs showed severe watery diarrhea and/or vomiting and mainly, diffuse, severe atrophic enteritis, with mild to moderate cytoplasmic vacuolation of the enteroctyes lining the atrophied villous epithelium. By IF, PDCoV antigens were evident in villous or crypt epithelial cells. No PDCoV antigen-positive, small and large intestinal villous or crypt epithelial cells, of which cytoplasm was also either vacuolated or morphologically normal, showed positive TUNEL staining. In contrast, by double IF and TUNEL staining, most of the TUNEL-positive signals (apoptotic nuclear fragmentation) were found in PDCoV antigen-positive LLC-PK and ST cells that also showed cytopathic effects, such as cell rounding, detachment and clumping in clusters. Secondary annexin V/propidium iodide (PI) staining revealed increased numbers of annexin V- or PI-positive LLC-PK and ST cells at 21h after inoculation, compared to the negative controls. Thus, PDCoV does not induce apoptosis in the infected intestinal enterocytes in vivo, but in two infected cell lines of swine origin, LLC-PK and ST cells.

Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

The human CLCA4 (chloride channel regulator, calcium-activated) modulates the intestinal phenotype of cystic fibrosis (CF) patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

Loss of NHE8 expression impairs intestinal mucosal integrity.

The newest member of the Na(+)/H(+) exchanger (NHE) family, NHE8, is abundantly expressed at the apical membrane of the intestinal epithelia. We previously reported that mucin 2 expression was significantly decreased in the colon in NHE8(-/-) mice, suggesting that NHE8 is involved in intestinal mucosal protection. In this study, we further evaluated the role of NHE8 in intestinal epithelial protection after dextran sodium sulfate (DSS) challenge. Compared with wild-type mice, NHE8(-/-) mice have increased bacterial adhesion and inflammation, especially in the distal colon. NHE8(-/-) mice are also susceptible to DSS treatment. Real-time PCR detected a remarkable increase in the expression of IL-1β, IL-6, TNF-α, and IL-4 in DSS-treated NHE8(-/-) mice compared with DSS-treated wild-type littermates. Immunohistochemistry showed a disorganized epithelial layer in the colon of NHE8(-/-) mice. Periodic acid-Schiff staining showed a reduction in the number of mature goblet cells and the area of the goblet cell theca in NHE8(-/-) mice. Phyloxine/tartrazine staining revealed a decrease in functional Paneth cell population in the NHE8(-/-) small intestinal crypt. The expression of enteric defensins was also decreased in NHE8(-/-) mice. The reduced mucin production in goblet cells and antimicrobial peptides production in Paneth cells lead to disruption of the intestinal mucosa protection. Therefore, NHE8 may be involved in the establishment of intestinal mucosal integrity by regulating the functions of goblet and Paneth cells.

The Viral Mimetic Polyinosinic:Polycytidylic Acid Alters the Growth Characteristics of Small Intestinal and Colonic Crypt Cultures.

Background & AimsThe intestinal epithelium is the first line of defense against enteric pathogens. We investigated the response of small intestinal and colonic crypt cultures to a panel of toll-like receptor ligands to assess the impact of microbial pattern recognition on epithelial growth.MethodsPrimary murine jejunal enteroids and colonoids were cultured with lipopeptide Pam3CSK4, lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (Poly I:C) for 4 to 6 days. Surface area, budding and survival were assessed. Proliferation and numbers of lysozyme positive cells were quantified by flow cytometry. Gene expression was assessed by Nanostring and qRT-PCR.ResultsExposure to Pam3CSK4 and LPS had minimal impact on either enteroids or colonoids. In contrast, Poly I:C increased the surface area of enteroids, while colonoids demonstrated decreased budding. Survival was decreased by Poly I:C in enteroids but not in colonoids. Both enteroids and colonoids exhibited upregulated gene expression of chemokines, but these were increased in magnitude in enteroids. Decreases in gene expression associated with epithelial differentiation and lysozyme positive cells were more apparent in enteroids than in colonoids. Baseline gene expression between enteroids and colonoids differed markedly in levels of stem cell and inflammatory markers. The changes in morphology induced by Poly I:C were mediated by the toll-like receptor adaptor molecule 1 (Ticam1) in enteroids but not in colonoids.ConclusionsPoly I:C alters the molecular program of epithelial cells and shifts from absorption and digestion towards defense and inflammation. Diversity of responses to microbial patterns in enteroids and colonoids may underlie differences in susceptibility to infection along the intestinal tract.

The Interplay between Wnt Mediated Expansion and Negative Regulation of Growth Promotes Robust Intestinal Crypt Structure and Homeostasis.

The epithelium of the small intestinal crypt, which has a vital role in protecting the underlying tissue from the harsh intestinal environment, is completely renewed every 4–5 days by a small pool of stem cells at the base of each crypt. How is this renewal controlled and homeostasis maintained, particularly given the rapid nature of this process? Here, based on the recent observations from in vitro “mini gut” studies, we use a hybrid stochastic model of the crypt to investigate how exogenous niche signaling (from Wnt and BMP) combines with auto-regulation to promote homeostasis. This model builds on the sub-cellular element method to account for the three-dimensional structure of the crypt, external regulation by Wnt and BMP, internal regulation by Notch signaling, as well as regulation by internally generated diffusible signals. Results show that Paneth cell derived Wnt signals, which have been observed experimentally to sustain crypts in cultured organs, have a dramatically different influence on niche dynamics than does mesenchyme derived Wnt. While this signaling can indeed act as a redundant backup to the exogenous gradient, it introduces a positive feedback that destabilizes the niche and causes its uncontrolled expansion. We find that in this setting, BMP has a critical role in constraining this expansion, consistent with observations that its removal leads to crypt fission. Further results also point to a new hypothesis for the role of Ephrin mediated motility of Paneth cells, specifically that it is required to constrain niche expansion and maintain the crypt’s spatial structure. Combined, these provide an alternative view of crypt homeostasis where the niche is in a constant state of expansion and the spatial structure of the crypt arises as a balance between this expansion and the action of various sources of negative regulation that hold it in check.

Bitter taste receptor mTas2r105 is expressed in small intestinal villus and crypts

The small intestine is the most important digestion and absorption organ in the body. Taste receptors and taste signal transduction cascades were detected in a variety of non-lingual tissues including testis, kidney, nasal cavity, lung, heart and gastrointestinal (GI) tract. Though the expression of bitter taste receptors and taste signal transduction cascades has been reported in the gut for a decade, the evidence revealing the expression of Tas2rs in the gut remain unbelievable. Here, the amplification of 35 bitter taste receptors from small intestine cDNA revealed that all transcripts are present in duodenum, jejunum and ileum, except Tas2r117. In addition, Tas2Rs and taste-related signaling transduction cascades are also observed in mouse small intestine including duodenum, jejunum and ileum by RT-PCR and Western Blot. On the other hand, three types of transgenic system were used to investigate the expression of the bitter taste receptor Tas2r105 in mouse intestine (Tas2r105-GFP/Cre, Tas2r105-GFP/Cre-DTA and Tas2r105-GFP/Cre-LacZ). With the bitter taste receptor mTas2r105 transgenic mice, the expression of mTas2r105 is showed in the villus and crypts of small intestine. mTas2r105 positive cells are also observed at the connective tissue of villus. DTA expression in mTas2r105 + cells completely ablate the expression of mTas2r105 in intestinal epithelia, but did not ablate mTas1r3 expression in intestine epithelia. LacZ staining further reveals that bitter taste receptor mTas2r105 is expressed in crypt base cells.

Microarray profiling to analyze the effect of Snai1 loss in mouse intestinal epithelium.

Epithelial stem cells from a variety of tissues have been shown to express genes linked to mesenchymal cell states. The Snail family of transcriptional factors has long been regarded as a marker of mesenchymal cells, however recent studies have indicated an involvement in regulation of epithelial stem cell populations. Snai1 is expressed in the stem cell population found at the base of the mouse small intestinal crypt that is responsible for generating all differentiated cell types of the intestinal epithelium. We utilized an inducible Cre recombinase approach in the intestinal epithelium combined with a conditional floxed Snai1 allele to induce knockout of gene function in the stem cell population. Loss of Snai1 resulted in loss of crypt base columnar cells and a failure to induce a proliferative response following radiation damage. We induced Snai1 loss in cultured organoids that had been derived from epithelial cells and compared gene expression to organoids with functional Snai1. Here we describe in detail the methods for generation of knockout organoids and analysis of microarray data that has been deposited in Gene Expression Omnibus (GEO):GSE65005.

Overlapping DNA methylation dynamics in mouse intestinal cell differentiation and early stages of malignant progression.

Mouse models of intestinal crypt cell differentiation and tumorigenesis have been used to characterize the molecular mechanisms underlying both processes. DNA methylation is a key epigenetic mark and plays an important role in cell identity and differentiation programs and cancer. To get insights into the dynamics of cell differentiation and malignant transformation we have compared the DNA methylation profiles along the mouse small intestine crypt and early stages of tumorigenesis. Genome-scale analysis of DNA methylation together with microarray gene expression have been applied to compare intestinal crypt stem cells (EphB2high), differentiated cells (EphB2negative), ApcMin/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Compared with late stages, small intestine crypt differentiation and early stages of tumorigenesis display few and relatively small changes in DNA methylation. Hypermethylated loci are largely shared by the two processes and affect the proximities of promoter and enhancer regions, with enrichment in genes associated with the intestinal stem cell signature and the PRC2 complex. The hypermethylation is progressive, with minute levels in differentiated cells, as compared with intestinal stem cells, and reaching full methylation in advanced stages. Hypomethylation shows different signatures in differentiation and cancer and is already present in the non-tumor tissue adjacent to the adenomas in ApcMin/+ mice, but at lower levels than advanced cancers. This study provides a reference framework to decipher the mechanisms driving mouse intestinal tumorigenesis and also the human counterpart.

Interleukin-22 directly regulates intestinal stem cell regeneration after tissue damage (MUC8P.722)

Abstract Little is known about regulation of the intestinal stem cell (ISC) compartment after injury. We hypothesized that IL-22 could promote intestinal regeneration by either acting on ISCs or by acting on Paneth cells (PCs) making up the ISC niche. Utilizing an in vitro regeneration model, we found that organoids derived from murine small intestine (SI) and large intestine (LI) crypts demonstrated substantially increased size after culture with IL-22. Innate lymphoid cell co-culture also increased organoid size in an IL-22-dependent fashion. IL-22 expanded the Lgr5+ ISC pool within SI organoids, activated STAT3 phosphorylation in Lgr5+ cells, and increased size of organoids cultured from purified SI ISCs. Using a murine allogeneic bone marrow transplant (BMT) model, we found that IL-22 administration led to reduced intestinal graft vs. host disease pathology, increased maintenance of Lgr5+ ISCs, and significantly greater ISC proliferation. ISC protection post-BMT was not due to niche augmentation, as PC numbers, PC-derived growth factors (EGF, Wnt3), and stroma-derived growth factors (R-spondin3) were all unchanged after IL-22 administration. IL-22 receptor (IL-22R) staining further indicated a direct effect of IL-22 on ISCs rather than the niche, as PCs were found to express very little IL-22R and demonstrated no evidence of STAT3 phosphorylation upon in vitro culture. In summary, we found that IL-22 regulates epithelial regeneration after tissue damage by acting directly on ISCs.

New developments in goblet cell mucus secretion and function.

Goblet cells and their main secretory product, mucus, have long been poorly appreciated; however, recent discoveries have changed this and placed these cells at the center stage of our understanding of mucosal biology and the immunology of the intestinal tract. The mucus system differs substantially between the small and large intestine, although it is built around MUC2 mucin polymers in both cases. Furthermore, that goblet cells and the regulation of their secretion also differ between these two parts of the intestine is of fundamental importance for a better understanding of mucosal immunology. There are several types of goblet cell that can be delineated based on their location and function. The surface colonic goblet cells secrete continuously to maintain the inner mucus layer, whereas goblet cells of the colonic and small intestinal crypts secrete upon stimulation, for example, after endocytosis or in response to acetyl choline. However, despite much progress in recent years, our understanding of goblet cell function and regulation is still in its infancy.

255 High Unsaturated Fat Diets and Unsaturated Visceral Fat Worsen Acute Pancreatitis (AP) Outcomes At Lower Body Mass Index (BMI)

Background & Aims: Environmental enteropathy (EE) is a subclinical intestinal condition highly prevalent in lowand middle-income countries characterized, in part, by malabsorption, intestinal villous atrophy, and crypt hypertrophy. The pathogenesis of EE remains unclear, however supplementation with folate (a key source of one carbon units for DNA methylation) is an effective adjunct therapy for tropical sprue, i.e., persistent diarrhea on a background of EE. To determine the extent to which methyl donor deficiency (MDD) provokes features of EE in mice, we evaluated mechanistic links between dietary methyl donor deficiency and intestinal crypt hypertrophy in mice and in mouse small intestinal crypt cultures (enteroids). Methods: We randomized dams to a standard diet or an isocaloric MDD diet lacking folate, choline, and betaine when pups were 10-days-old. We then randomized weanlings to their dams' diet on day of life 21. Mice were sacrificed and the jejunum was harvested at 6 weeks of age for both histology (n=6/group) and generation of enteroids. Results: Histological comparisons of the jejunum revealed longer crypts in MDD versus control mice, moreso in the distal (85.6 +/13.7 μm vs. 69.3 +/7.2 μm; P<0.0001), vs. proximal (88.8 +/17 μm vs 80.7 +/10.7 μm; P=0.04) portions of the small intestine. Enteroids from control and MDD mice were both viable in standard minigut media; however, all enteroids maintained in MDD media displayed alterations in crypt morphology and decreased epithelial proliferation. Enteroid crypt neck width was 1.3-fold greater in standard vs. MDD media (P<0.001). The number of crypt buds per enteroid was 1.6-fold higher in standard media vs. MDD media (p = 0.0007). The ratio of EdU-positive, or proliferating, cells to Hoechst-positive cells was 2.2-fold higher in standard media vs. MDD media (P<0.0001). Qualitatively, enteroids in MDD media displayed longer crypt domains vs. enteroids in standard media. Conclusions: Complementary in vivo and in vitro findings of altered small intestinal crypt in the setting of methyl donor deficiency suggest that methyl donor deficiency plays a role in the pathogenesis of environmental enteropathy. Further studies are needed to determine whether prolonged methyl donor deficiency promotes epigenetic changes of intestinal stem cells and whether methyl donor supplementation prevents or reverses these changes to promote intestinal epithelial homeostasis in global settings of poverty.

The Zinc Transporter ZnT2 is Necessary for Stabilization of Granule Contents in Intestinal Paneth Cell Granules

Paneth cells (PC) are secretory cells in small intestinal crypts that play a critical role in self‐protection against luminal pathogens. Defects in PC function are associated with necrotizing enterocolitis and Crohn's disease. PCs release granules containing a host of antimicrobial peptides and a large amount of zinc (Zn). Zn prevents degradation of α ‐defensins and stabilizes lysozyme in its monomeric active conformation. How Zn accumulates in PC granules is not understood. We used wild‐type (WT) and ZnT2‐null (KO) mice to determine the role of ZnT2 in PC function. ZnT2 was expressed in crypts and localized to PC granules by immunofluorescence and sucrose gradient fractionation. Studies using the fluorescent Zn reporter ZP1 indicated that KO mice were unable to accumulate Zn into granules. While no morphologic abnormalities were observed in the intestines of KO mice, a significantly greater number of PCs were degranulated compared with PCs in WT mice (p&lt;0.01), and PCs in KO mice had profound defects in sub‐cellular organization. In response to saline perfusion, ileal segments from KO mice retained less overall (p&lt;0.01), and significantly more dimerized (less active) lysozyme (p&lt;0.05), but released more lysozyme into the lumen (p&lt;0.05). These data indicate that ZnT2 imports Zn into PC granules, and the inability to do so destabilizes granules and Zn‐requiring granule proteins and leads to defects in coordinated granule secretion, which may put an individual at risk for infection and inflammatory illness.