Intestinal Epithelial Crypt Research Articles

Protective effects of CXCR3/HO‑1 gene‑modified BMMSCs on damaged intestinal epithelial cells: Role of the p38‑MAPK signaling pathway.

The purpose of the present study was to investigate whether bone marrow mesenchymal stem cells (BMMSCs) modified by CXC-chemokine receptor type 3 (CXCR3) and heme oxygenase-1 (HO-1) genes can repair damaged intestinal epithelial cells in vitro, and the role of the p38 mitogen-activated protein kinase (p38-MAPK) pathway in this process. A model of intestinal epithelial crypt cell line-6 (IEC-6) damage was created, and BMMSCs were transfected with either the CXCR3 and/or HO-1 gene in vitro. There were nine experimental groups in which the damaged IEC-6 cells were co-cultured with differentially-treated BMMSCs and lymphocytes for 24 h. Reverse transcription-quantitative polymerase chain reaction analysis, immunohistochemistry and a western blot analysis were performed to detect stem cell transfection, the repair of damaged intestinal epithelial cells and the expression of related molecules in the P38-MAPK pathway, respectively. Crystal violet staining and live cell imaging were used to detect the chemotaxis of BMMSCs. Flow cytometry was used to detect T lymphocyte activity and the surface markers expressed on BMMSCs. An ELISA was used to quantify cytokine production. The adenovirus (Ad)-CXCR3/MSCs exhibited the characteristics of stem cells and exhibited chemotaxis. The Ad-CXCR3/MSCs and Ad-(CXCR3 + HO)/MSCs exhibited increased expression of tight junction protein zonula occludens-1 (ZO-1) and anti-proliferating cell nuclear antigen in the damaged IEC-6 cells, and apoptosis of the damaged IEC-6 cells was decreased. BMMSCs inhibited the phosphorylation of p38, in addition to downstream molecules of the p38MAPK signaling pathway. The Ad-CXCR3/MSCs and Ad-(CXCR3 + HO)/MSCs exhibited significantly decreased expression levels of downstream molecules, including phosphorylated (p)-p38, p-activated transcription factor 2, p-C/EBP homologous protein-10, and p-myocyte enhancer factor 2C, and target molecules (e.g., apoptotic bodies). The effects of Ad-(CXCR3 + HO)/MSCs on the repair of the damaged intestinal tract and inhibition of the p38-MAPK pathway was more marked than those in other groups on day 7 post-surgery in the rejection model for small bowel transplantation. BMMSCs modified by the CXCR3 and HO-1 genes exhibited superior ability to repair damaged intestinal epithelial cells and served this role via the p38-MAPK pathway.

The In Vitro Protective Role of Bovine Lactoferrin on Intestinal Epithelial Barrier

The intestinal epithelial barrier plays a key protective role in the gut lumen. Bovine lactoferrin (bLF) has been reported to improve the intestinal epithelial barrier function, but its impact on tight junction (TJ) proteins has been rarely described. Human intestinal epithelial crypt cells (HIECs) were more similar to those in the human small intestine, compared with the well-established Caco-2 cells. Accordingly, both HIECs and Caco-2 cells were investigated in this study to determine the effects of bioactive protein bLF on their growth promotion and intestinal barrier function. The results showed that bLF promoted cell growth and arrested cell-cycle progression at the G2/M-phase. Moreover, bLF decreased paracellular permeability and increased alkaline phosphatase activity and transepithelial electrical resistance, strengthening barrier function. Immunofluorescence, western blot and quantitative real-time polymerase chain reaction revealed that bLF significantly increased the expression of three tight junction proteins—claudin-1, occludin, and ZO-1—at both the mRNA and protein levels, and consequently strengthened the barrier function of the two cell models. bLF in general showed higher activity in Caco-2 cells, however, HIECs also exhibited desired responses to barrier function. Therefore, bLF may be incorporated into functional foods for treatment of inflammatory bowel diseases which are caused by loss of barrier integrity.

Mist1 Expression Is Required for Paneth Cell Maturation.

BackgroundPaneth cells are professional secretory cells found within the small intestinal crypt epithelium. Although their role as part of the innate immune complex providing antimicrobial secretory products is well-known, the mechanisms that control secretory capacity are not well-understood. MIST1 is a scaling factor that is thought to control secretory capacity of exocrine cells.MethodsMist1+/+ and Mist1–/– mice were used to evaluate the function of MIST1 in small intestinal Paneth cells. We used histologic and immunofluorescence staining to evaluate small intestinal tissue for proliferation and lineage allocation. Total RNA was isolated to evaluate gene expression. Enteroid culture was used to evaluate the impact of the absence of MIST1 expression on intestinal stem cell function.ResultsAbsence of MIST1 resulted in increased numbers of Paneth cells exhibiting an intermediate cell phenotype but otherwise did not alter overall epithelial cell lineage allocation. Muc2 and lysozyme staining confirmed the presence of intermediate cells at the crypt base of Mist1–/– mice. These changes were not associated with changes in mRNA expression of transcription factors associated with lineage allocation, and they were not abrogated by inhibition of Notch signaling. However, the absence of MIST1 expression was associated with alterations in Paneth cell morphology including decreased granule size and distended rough endoplasmic reticulum. Absence of MIST1 was associated with increased budding of enteroid cultures; however, there was no evidence of increased intestinal stem cell numbers in vivo.ConclusionsMIST1 plays an important role in organization of the Paneth cell secretory apparatus and managing endoplasmic reticulum stress. This role occurs downstream of Paneth cell lineage allocation.

FAK Alleviates Radiation-Induced Rectal Injury Through AKT Signaling Pathway

Rectum is a dose-limiting organ in pelvic tumors radiotherapy. The assessing of radiosensitivity is good to improve individualized radiotherapy. Our previous study has shown increased FAK (focal adhesion kinase, PTK2) expression was related with radioresistance of intestinal epithelial cells. However, its molecular mechanism remains unclear. Here, we investigated the effect of FAK in radiation-induced rectal injury. Peripheral blood samples of patients with colorectal cancer were collected prior to radiotherapy. Differentially expressed genes and copy number variations (CNV) were analyzed by microarray in blood samples. The CTCAE version 3.0 toxicity grades was used assess the acute rectal injury. The intestinal epithelial cells are mostly the main targets for radiation damage. A normal human intestinal epithelial crypt cell line (HIEC) was used. The radiosensitivity of HIEC cells were detected by colony formation assay and flow cytometry. Further studies on AKT signaling-related and apoptotic-related proteins were assessed by western blot. The inhibitors (VS-4718 and MK2206 2HCL) or siRNAs of FAK or AKT were used in the process of above detection. The rectum of C57BL/6N mice were X-irradiated locally with a single dose of 25Gy, and following experiments including hematoxylin-eosin (HE) staining, real-time PCR and immunohistochemistry (IHC). In addition, the effect of FAK-AKT on radiation-induced injury was investigated by the administration of AKT inhibitor after irradiation. FAK mRNA was highly expressed in the mild rectal injury patient samples compared with the severe injury patient samples. In addition, the CNV amplification was located on chromosome 8p24.3, which was related closely with FAK. Further study demonstrated FAK-silenced HIEC cells were more radiosensitive by colony survival and apoptosis assay. The expression of γH2AX involved in DNA damage and repair increased either at 6 h or at 24 h after irradiation treatment. Further functional assays revealed that depressed AKT, increased cytochrome C and cleaved caspase-3 in FAK-silenced HIEC cells. In vivo , radiation-induced apoptosis of crypt cells are under analyzed with or without AKT inhibition. The effect of FAK was known in cancer cells. In current study, our data showed that FAK expression was consistent mild radiation-induced rectal injury. These findings demonstrated that FAK reduced the radiosensitivity of intestinal epithelial cells, which may be related with AKT signaling pathway and apoptotic-related proteins. In summary, further studies based on better understanding of FAK may be benefit to predict and prevent radiation-induced normal tissue injury.

FAK alleviates radiation-induced rectal injury by decreasing apoptosis

Radiation-induced rectal injury is closely related with radiotherapy efficiency. Here, we investigated the effect of focal adhesion kinase (FAK) in radiation-induced rectal injury. Peripheral blood samples of patients with rectal cancer were collected prior to radiotherapy. Differentially expressed genes and copy number variations (CNVs) were analyzed by microarray analysis. The CTCAE v3.0 toxicity grades were used to assess acute rectal injury. The radiosensitivity of human intestinal epithelial crypt (HIEC) cells were assayed by colony formation, mitochondrial membrane potential, flow cytometry and western blotting. The rectums of C57BL/6 mice were X-irradiated locally with a single dose of 15 Gy. The effect of FAK on radiation-induced injury was investigated by hematoxylin-eosin (H&E) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR). FAK mRNA level was inversely correlated with rectal injury severity in patient samples. A CNV amplification located on chromosome 8 was closely related with FAK. Further functional assays revealed increased levels of γH2AX expression and apoptosis-related proteins in FAK-silenced HIEC cells. The ratio of TUNEL, cl-caspase-3, cyto-c and bax/bcl-2 expression in the rectum mucosa treated with a FAK inhibitor increased significantly. These results demonstrated that FAK reduced radiation-induced rectal injury by decreasing apoptosis.

Insulin/IGF-1 enhances intestinal epithelial crypt proliferation through PI3K/Akt, and not ERK signaling in obese humans.

The intestinal epithelium is continuously regenerated through proliferation and differentiation of stem cells located in the intestinal crypts. Obesity affects this process and results in greater stem cell proliferation and altered tissue growth and function. Obesity-induced high levels of insulin and insulin-like growth factor-1 in the stem cell niche are found to impact proliferation in rodents indicating that insulin and insulin-like growth factor-1 receptors may play a role in modulating intestinal epithelial stem cell proliferation. To determine whether insulin or insulin-like growth factor-1 can induce proliferation in human intestinal epithelial stem cells, and if two downstream insulin and insulin-like growth factor-1 receptor signaling pathways, PI3K/Akt and ERK, are involved, we used primary small intestinal epithelial crypts isolated from obese humans and investigated (1) the effect of insulin or insulin-like growth factor-1 on crypt proliferation, and (2) the effect of insulin and insulin-like growth factor-1 signaling inhibitors on insulin or insulin-like growth factor-1-induced proliferation. We found that insulin and insulin-like growth factor-1 enhanced the proliferation of crypt cells, including intestinal epithelial stem cells. Inhibition of the PI3K/Akt pathway attenuated insulin and insulin-like growth factor-1-induced proliferation, but inhibition of the ERK pathway had no effect. These results suggest that the classical metabolic PI3K pathway and not the canonical proliferation ERK pathway is involved in the insulin/insulin-like growth factor-1-induced increase in crypt proliferation in obese humans, which may contribute to abnormal tissue renewal and function. Impact statement This study investigates if insulin or insulin-like growth factor-1 (IGF-1) induces intestinal epithelial proliferation in humans, and if insulin and IGF-1 receptor signaling is involved in this process in obesity. Although obesity-induced high levels of insulin and IGF-1 in the stem cell niche are found to impact the proliferation of intestinal epithelial stem cells in rodents, we are the first to investigate this effect in humans. We found that insulin and IGF-1 enhanced the proliferation of intestinal crypts (including stem cells and other crypt cells) isolated from obese humans, and PI3K/Akt, and not ERK signaling was involved in insulin or IGF-1-induced proliferation. The imbalance in signaling between PI3K/Akt and ERK pathways may point to a pathway-specific impairment in insulin/IGF-1 receptor signaling. We propose that this may contribute to reciprocal relationships between insulin/IGF-1 receptor resistance and intestinal epithelial proliferation that leads to abnormal tissue renewal and function.

Insulin/IGF‐1 Enhances Intestinal Epithelial Crypt Proliferation through PI3K/Akt, and Not ERK Signaling in Obese Humans

The intestinal epithelium plays important roles in nutrient absorption, satiety hormone release and immune barrier function. In order to ensure proper tissue actions are maintained, the cells are continuously regenerated through proliferation and differentiation of stem cells located in the intestinal crypts. Obesity affects this process and results in greater stem cell proliferation and abnormal tissue growth and function. Obesity‐induced high levels of insulin/insulin‐like growth factor‐1 (IGF‐1) in the stem cell niche are found to impact the changes in proliferation in rodents indicating that insulin/IGF‐1 receptors may play a role in modulating intestinal epithelial stem cell proliferation. Insulin receptor resistance is a hallmark of obesity that can be driven by an imbalance between or abnormal signaling within two downstream signaling pathways, PI3K and ERK, in non‐intestinal epithelial tissues. To determine whether insulin/IGF‐1 can induce proliferation in human intestinal epithelial stem cells and if PI3K or ERK pathways are involved, we used primary small intestinal epithelial crypts and investigated 1) the effect of insulin/IGF‐1 on crypt proliferation, and 2) the effect of insulin/IGF‐1 signaling inhibitors (i.e. PI3K/Akt pathway inhibitor Wortmannin and ERK pathway inhibitor PD98059) on insulin/IGF‐1‐induced crypt proliferation. We found that insulin/IGF‐1 enhanced crypt proliferation in obese humans. Inhibition of PI3K/Akt pathway attenuated insulin/IGF‐1‐induced crypt proliferation, but inhibition of ERK pathway had no effect. These results suggest that the classical metabolic PI3K pathway and not the canonical proliferation ERK pathway is involved in insulin/IGF‐1‐induced increase in crypt proliferation in obese humans. Pathway‐specific impairments in insulin/IGF‐1 signaling may contribute to reciprocal relationships between insulin/IGF‐1 resistance and intestinal epithelial stem cell proliferation and differentiation that fosters tissue dysfunction.Support or Funding InformationThis work was supported by USDA Hatch (to MJD) ILLU‐538‐926.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glucose stimulates intestinal epithelial crypt proliferation by modulating cellular energy metabolism.

The intestinal epithelium plays an essential role in nutrient absorption, hormone release, and barrier function. Maintenance of the epithelium is driven by continuous cell renewal by stem cells located in the intestinal crypts. The amount and type of diet influence this process and result in changes in the size and cellular make-up of the tissue. The mechanism underlying the nutrient-driven changes in proliferation is not known, but may involve a shift in intracellular metabolism that allows for more nutrients to be used to manufacture new cells. We hypothesized that nutrient availability drives changes in cellular energy metabolism of small intestinal epithelial crypts that could contribute to increases in crypt proliferation. We utilized primary small intestinal epithelial crypts from C57BL/6J mice to study (1) the effect of glucose on crypt proliferation and (2) the effect of glucose on crypt metabolism using an extracellular flux analyzer for real-time metabolic measurements. We found that glucose increased both crypt proliferation and glycolysis, and the glycolytic pathway inhibitor 2-deoxy-d-glucose (2-DG) attenuated glucose-induced crypt proliferation. Glucose did not enhance glucose oxidation, but did increase the maximum mitochondrial respiratory capacity, which may contribute to glucose-induced increases in proliferation. Glucose activated Akt/HIF-1α signaling pathway, which might be at least in part responsible for glucose-induced glycolysis and cell proliferation. These results suggest that high glucose availability induces an increase in crypt proliferation by inducing an increase in glycolysis with no change in glucose oxidation.

LKB1-AMPK modulates nutrient-induced changes in the mode of division of intestinal epithelial crypt cells in mice.

Nutrient availability influences intestinal epithelial stem cell proliferation and tissue growth. Increases in food result in a greater number of epithelial cells, villi height and crypt depth. We investigated whether this nutrient-driven expansion of the tissue is the result of a change in the mode of intestinal epithelial stem cell division and if LKB1-AMPK signaling plays a role. We utilized invivo and invitro experiments to test this hypothesis. C57BL/6J mice were separated into four groups and fed varying amounts of chow for 18 h: (1) ad libitum, (2) 50% of their average daily intake (3) fasted or (4) fasted for 12 h and refed. Mice were sacrificed, intestinal sections excised and immunohistochemically processed to determine the mitotic spindle orientation. Epithelial organoids invitro were treated with no (0 mM), low (5 mM) or high (20 mM) amounts of glucose with or without an activator (Metformin) or inhibitor (Compound C) of LKB1-AMPK signaling. Cells were then processed to determine the mode of stem cell division. Fasted mice show a greater % of asymmetrically dividing cells compared with the other feeding groups. Organoids incubated with 0 mM glucose resulted in a greater % of asymmetrically dividing cells compared with the low or high-glucose conditions. In addition, LKB1-AMPK activation attenuated the % of symmetric division normally seen in high-glucose conditions. In contrast, LKB1-AMPK inhibition attenuated the % of asymmetric division normally seen in no glucose conditions. These data suggest that nutrient availability dictates the mode of division and that LKB1-AMPK mediates this nutrient-driven effect on intestinal epithelial stem cell proliferation. Impact statement The underlying cell biology of changes in the polarity of mitotic spindles and its relevance to tissue growth is a new concept and, thus, these data provide novel findings to begin to explain how this process contributes to the regeneration and growth of tissues. We find that short-term changes in food intake invivo or glucose availability invitro dictate the mode of division of crypt cells. In addition, we find that LKB1-AMPK signaling modulates the glucose-induced changes in the mode of division invitro. Identifying mechanisms involved in the mode of division may provide new targets to control tissue growth.

Mutual reinforcement between telomere capping and canonical Wnt signalling in the intestinal stem cell niche

Critical telomere shortening (for example, secondary to partial telomerase deficiency in the rare disease dyskeratosis congenita) causes tissue pathology, but underlying mechanisms are not fully understood. Mice lacking telomerase (for example, mTR−/− telomerase RNA template mutants) provide a model for investigating pathogenesis. In such mice, after several generations of telomerase deficiency telomeres shorten to the point of uncapping, causing defects most pronounced in high-turnover tissues including intestinal epithelium. Here we show that late-generation mTR−/− mutants experience marked downregulation of Wnt pathway genes in intestinal crypt epithelia, including crypt base columnar stem cells and Paneth cells, and in underlying stroma. The importance of these changes was revealed by rescue of crypt apoptosis and Wnt pathway gene expression upon treatment with Wnt pathway agonists. Rescue was associated with reduced telomere-dysfunction-induced foci and anaphase bridges, indicating improved telomere capping. Thus a mutually reinforcing feedback loop exists between telomere capping and Wnt signalling, and telomere capping can be impacted by extracellular cues in a fashion independent of telomerase.

Sox9 transcriptionally regulates Wnt signaling in intestinal epithelial stem cells in hypomethylated crypts in the diabetic state

BackgroundDistinctive structures called crypts harbor intestinal epithelial stem cells (IESCs) which generate progenitor and terminally differentiated cells in the intestinal epithelium. Mammalian IESCs and their daughter cells require the participation of DNA methylation and the transcription factor Sox9 for proliferation and differentiation. However, the association between Sox9 and DNA methylation in this process remains elusive.MethodsThe DNA methylation of small intestinal epithelial crypts in db/db mice was detected via combining methylated DNA immunoprecipitation with microarray hybridization. DNA methylation of Sox9 promoter in crypts and IESCs was validated using bisulfite sequence analysis. The target sequence of the transcription factor Sox9 in IESCs was investigated via chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq).ResultsIncreased Sox9 expression is accompanied by the loss of methylation in its promoter in IESCs. Sox9 targets the enhancers of the Wnt signaling pathway-related genes. Sox9 predominantly acts as a transcriptional activator at proximal enhancers of Wnt4, Tab2, Sox4, and Fzd8, but also functions as a potential transcriptional inhibitor at a distant enhancer of Cdk1. Lack of Sox9 transcriptional activation in specific repressors of the Wnt signaling pathway leads to the loss of intrinsic inhibitory action and ultimately produces overactivation of this pathway in db/db mice.ConclusionsOur study sheds light on the connections among DNA methylation, transcription factor modulation, and Wnt signaling in IESCs in the diabetic state. Hypomethylation in the Sox9 promoter is correlated to increased Sox9 expression in db/db IESCs. Although there is increased expression of Sox9 in db/db IESCs, the loss of Sox9 transcriptional activation in specific repressors of the Wnt signaling pathway might result in abnormalities in this pathway.

Clr-a: A Novel Immune-Related C-Type Lectin-like Molecule Exclusively Expressed by Mouse Gut Epithelium.

The mouse gut epithelium represents a constitutively challenged environment keeping intestinal commensal microbiota at bay and defending against invading enteric pathogens. The complex immunoregulatory network of the epithelial barrier surveillance also involves NK gene complex (NKC)-encoded C-type lectin-like molecules such as NKG2D and Nkrp1 receptors. To our knowledge, in this study, we report the first characterization of the orphan C-type lectin-like molecule Clr-a encoded by the Clec2e gene in the mouse NKC. Screening of a panel of mouse tissues revealed that Clec2e transcripts are restricted to the gastrointestinal tract. Using Clr-a-specific mAb, we characterize Clr-a as a disulfide-linked homodimeric cell surface glycoprotein. Of note, a substantial fraction of Clr-a molecules are retained intracellularly, and analyses of Clr-a/Clr-f hybrids attribute intracellular retention to both the stalk region and parts of the cytoplasmic domain. Combining quantitative PCR analyses with immunofluorescence studies revealed exclusive expression of Clr-a by intestinal epithelial cells and crypt cells throughout the gut. Challenge with polyinosinic-polycytidylic acid results in a rapid and strong downregulation of intestinal Clr-a expression in contrast to the upregulation of Clr-f, a close relative of Clr-a, that also is specifically expressed by the intestinal epithelium and acts as a ligand of the inhibitory Nkrp1g receptor. Collectively, we characterize expression of the mouse NKC-encoded glycoprotein Clr-a as strictly associated with mouse intestinal epithelium. Downregulation upon polyinosinic-polycytidylic acid challenge and expression by crypt cells clearly distinguish Clr-a from the likewise intestinal epithelium-restricted Clr-f, pointing to a nonredundant function of these highly related C-type lectin-like molecules in the context of intestinal immunosurveillance.

Defective ATG16L1-mediated removal of IRE1α drives Crohn's disease-like ileitis.

ATG16L1T300A, a major risk polymorphism in Crohn's disease (CD), causes impaired autophagy, but it has remained unclear how this predisposes to CD. In this study, we report that mice with Atg16l1 deletion in intestinal epithelial cells (IECs) spontaneously develop transmural ileitis phenocopying ileal CD in an age-dependent manner, driven by the endoplasmic reticulum (ER) stress sensor IRE1α. IRE1α accumulates in Paneth cells of Atg16l1ΔIEC mice, and humans homozygous for ATG16L1T300A exhibit a corresponding increase of IRE1α in intestinal epithelial crypts. In contrast to a protective role of the IRE1β isoform, hyperactivated IRE1α also drives a similar ileitis developing earlier in life in Atg16l1;Xbp1ΔIEC mice, in which ER stress is induced by deletion of the unfolded protein response transcription factor XBP1. The selective autophagy receptor optineurin interacts with IRE1α, and optineurin deficiency amplifies IRE1α levels during ER stress. Furthermore, although dysbiosis of the ileal microbiota is present in Atg16l1;Xbp1ΔIEC mice as predicted from impaired Paneth cell antimicrobial function, such structural alteration of the microbiota does not trigger ileitis but, rather, aggravates dextran sodium sulfate-induced colitis. Hence, we conclude that defective autophagy in IECs may predispose to CD ileitis via impaired clearance of IRE1α aggregates during ER stress at this site.

Current advances in renewal mechanisms of intestinal epithelial cells along the crypt-villus axis

The small intestine is not only an organ for food digestion and nutrient absorption but also an integral part of the immune and endocrine systems. Differentiation of enterocytes plays an important role in the damage repair mechanism after immune response elicitation by the intestine, for restoring the intestinal barrier function and homeostasis. Recently, research related to self-renewal, differentiation, and regulation of the intestinal epithelial crypt stem cells has been developing rapidly. In this review, we attempt to sum up recent studies related to the expression pattern of genes and proteins along the crypt-villus axis, and the effect of signaling pathways, transcription factors, and epigenetic modifications on the differentiation of crypt stem cells. We also examine the mechanisms by which nutritional factors influence the differentiation and damage repair processes of the intestinal epithelial cells. This review could provide valuable theoretical framework in nutritional and pharmacological aspects and guidance for intervention and treatment of intestinal damage and related diseases.

Goblet cell depletion in small intestinal villous and crypt epithelium of conventional nursing and weaned pigs infected with porcine epidemic diarrhea virus

Intestinal goblet cells secret mucins to form mucus layers critical for maintaining the integrity of the intestinal epithelium. Porcine epidemic diarrhea virus (PEDV) causes watery diarrhea and high mortality of suckling pigs. PEDV mainly infects villous epithelial cells of the small intestine, and infected cells undergo acute, massive necrosis, followed by severe villous atrophy. Conventional 9-day-old nursing pigs [PEDV-inoculated (n=9); Mock (n=11)] and 26-day-old weaned [PEDV-inoculated (n=11); Mock (n=9)] were inoculated orally [8.9 log10 genomic equivalents/pig] with PEDV strain PC21A or mock. We used alcian blue or Periodic-Acid-Schiff staining for the detection of acidic or neutral mucin-secreting goblet cells in the small intestine. We demonstrated that PEDV infection of the nursing pigs at post-inoculation days (PIDs) 1–5 and weaned pigs at PIDs 3–5 led to depletion or significant reduction in the number of goblet cells (and also the number of villous goblet cells normalized by jejunal villous crypt height to crypt depth ratios) in the villi or crypts. These findings coincided with the development of intestinal villous atrophy. By immunohistochemistry, a few PEDV antigen-positive goblet cells were identified in the jejunal or ileal villous epithelium of the infected nursing or weaned pigs. During the early stages of PEDV infection, goblet cell mucins in the small intestine may be decreased, possibly leading to an impaired mucus layer and increased susceptibility to secondary enteric bacterial infection.

MiR-29a Regulates Radiosensitivity in Human Intestinal Cells by Targeting PTEN Gene.

Two major challenges encountered during radiotherapy for colorectal cancer (CRC) are radioresistance of tumor cells and damage to normal cells. An understanding of the mechanisms of radioresistance in CRC may lead to new strategies for overcoming obstacles to affective clinical therapy. In this study, the miR-29a expression was compared among four cell lines: the normal human intestinal epithelial crypt cell line, HIEC and three CRC cell lines, HT29, DLD-1 and HCT116. The roles of miR-29a in regulating cellular radiosensitivity were then investigated. The findings from this study showed that miR-29a mimic enhanced radioresistance in HIEC, HT29 and DLD-1 cells with low levels of intrinsic miR-29a. On the other hand, a miR-29a inhibitor significantly sensitized HCT116 cells with high levels of miR-29a after irradiation. Further studies indicated that PTEN was the direct functional target of miR-29a and was involved in radiosensitivity. MiR-29a could activate the PI3K/Akt signaling pathway through negatively regulated PTEN expression. In conclusion, miR-29a may regulate the radiosensitivity of intestinal cell lines by targeting the PTEN gene, which indicates miR-29a might serve as a novel approach to enhance radiosensitivity in CRC.

Canine Circovirus 1 (CaCV-1) and Canine Parvovirus 2 (CPV-2): Recurrent Dual Infections in a Papillon Breeding Colony.

Recurrent outbreaks of sudden death and bloody diarrhea were reported in March 2013 and February 2014 in a breeding colony of Papillon dogs. During the first outbreak, 1 adult dog and 2 eight-month-old puppies died. During the second outbreak, 2 ten-week-old puppies died. One puppy from the first outbreak and 2 puppies from the second outbreak were examined at necropsy. Histologically, all 3 puppies had severe segmental crypt necrosis of the small intestine and marked lymphoid follicle depletion in the spleen and Peyer's patches. Real-time (RT) polymerase chain reaction (PCR) demonstrated abundant canine parvovirus (CPV-2) DNA (Ct<15) in the affected small intestine, and immunohistochemistry detected large amounts of CPV-2 antigen in intestinal crypt epithelium and Kupffer cells but few positive macrophages in lymphoid organs. All puppies had marked sinusoidal histiocytosis and multifocal granulomatous inflammation in mesenteric lymph nodes and spleen, prompting additional RT-PCR testing for canine circovirus 1 (CaCV-1). Very high levels of CaCV-1 DNA (Ct<13) were detected in small intestine, lymph nodes, and spleen. In situ hybridization for CaCV-1 detected rare positive nuclei of regenerating crypt epithelium but abundant amounts of CaCV-1 nucleic acid in the cytoplasm and nuclei of histiocytes in all lymphoid tissues, including granulomatous inflammatory foci and hepatic Kupffer cells. Significant levels of CaCV-1 DNA were detected in blood and serum (Ct as low as 13) but not feces from 3 surviving dogs at 2 months or 1 year after the outbreak, respectively. We hypothesize that CPV-2 infection predisposed dogs to CaCV-1 infection and ultimately resulted in more severe clinical disease.

Intestinal Form of Rabbit Haemorrhagic Disease in Growing Rabbits (Oryctolagus cuniculus).

Rabbit Haemorrhagic Disease (RHD) is extremely acute highly fatal, contagious disease with mortality rates of 80-90% of the infected rabbits. RHD causes hepatic, intestinal and lymphoid necrosis with massive terminal intravascular coagulopathy. The etiological agent is a member of caliciviridae lagovirus, Rabbit Haemorrhagic Disease Virus (RHDV); it is a single stranded RNA, non-enveloped and replicates in the cytoplasm. In pathogenesis studies, the primary sites of replication were in the small intestinal crypt and villous epithelium, hepatocytes and splenic lymphocytes. Apoptosis (programmed cell death) has been reported as being a constant feature of the pathogenesis of RHD. This work was planned to study the lesions associated with RHDV in small intestine at different intervals. Eighteen growing New Zealand rabbits (Oryctolagus cuniculus) aged 2-3 months allotted into two equal groups: control group (non infected) and infected group in which rabbits were experimentally inoculated with rabbit hemorrhagic disease virus (RHDV) through the nostril. All animals were dissected at 24, 48 and 72 hrs post infection. Histopathological, histochemical, immunohistochemical and biochemical studies were done for small intestine.

Vitamin A Supplementation and Lipopolysaccharide‐Induced Inflammation in the Neonate — A Pilot Study in C57BL/6 Mice

Vitamin A (VA) is important in host defense against infections. Previous studies have suggested that retinoic acid, the active metabolite of VA, is critical in mucosal immunity and immune tolerance. However, studies are limited about how VA supplementation affects mucosal immune responses to inflammation in neonates. The objective of the study was to investigate the interactions between micronutrient (i.e., VA supplementation) and inflammation during the neonatal period, when intestinal epithelial crypts are formed and immune cell populations rapidly increase. We hypothesized there would be both independent effects of VA supplementation and lipopolysaccharide (LPS) treatment, as well as a potential modifying effect of VA supplementation on the LPS‐induced inflammatory response in neonates. A purified AIN‐93 VA‐marginal diet, containing 0.35 μg of retinol as retinyl palmitate per g diet, was fed to C57BL/6 dams from pregnancy until their pups (n=16) were euthanized on postnatal day (P) 14. Two doses of VA, 6 mg/kg of body weight, or canola oil as control, were administered orally on P12 and P13. LPS, 2 mg/kg of body weight, or phosphate‐buffered saline (PBS) as control, was injected intraperitoneally 3 h after the second dose of VA or oil on P13 to induce inflammation. Plasma and tissues were collected 18 h after LPS injection on P14. The small intestine samples were divided into upper and lower sections to determine differences between intestinal sections. Tissue total retinoid concentrations were determined by Ultra Performance Liquid Chromatography (UPLC). Cytokine analyses, including IFN‐γ, IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, KC/GRO, IL‐10, IL12p70, and TNF‐α, were performed on plasma and intestinal tissue homogenates using V‐PLEX validated assay kits. For statistical analysis, two‐way ANOVA was performed to examine two independent factors (i.e., VA supplementation and LPS‐induced inflammation), as well as their interactions. Differences among treatment groups were determined by Tukey's multiple comparison test. Pups with LPS‐induced inflammation lost 7.5% of body weight and had reduced stomach weight within the 18‐h period after LPS injection, while control pups gained 10.8% body weight. Total retinol concentrations in tissues, including liver, lung, and upper and lower small intestine, were significantly higher in VA‐supplemented as compared to oil‐treated pups. However, no significant difference was observed on tissue retinoid storage between LPS‐treated and control pups. Serum cytokine concentrations, including IFN‐γ, IL‐4, IL‐6, KC/GRO, IL‐10, IL12p70, and TNF‐α, were significantly higher in LPS‐treated pups regardless of VA treatment. Overall, cytokine concentrations were much higher in the intestine as compared to in serum, other than for IL‐10. For both upper and lower small intestine, the concentrations of IL‐1β and KC/GRO were higher in LPS‐treated pups and, interestingly, even higher in the VA‐supplemented pups under LPS‐induced inflammation. The results indicated that VA supplementation and LPS exert differential effects, but VA might also affect intestinal immune responses to LPS‐induced inflammation in neonates. Future studies are warranted to further investigate these interactions.

Nutrient‐Induced Metabolism Dictates Intestinal Epithelial Crypt Proliferation

The intestinal epithelium plays an essential role in nutrient absorption, hormone release and barrier function. Maintenance of the epithelium is driven by continuous cell renewal by stem cells located in the intestinal crypts. The amount and type of diet influence this process and result in changes in the size and cellular make‐up of the tissue. We have found that glucose induces an increase in the rate of proliferation in intestinal epithelial crypts in a concentration dependent manner. The mechanism underlying this nutrient‐driven change in crypt proliferation is not known, but may involve a shift in intracellular metabolism that allows for more nutrients to be used to manufacture new cells. We hypothesized that nutrient availability drives changes in cellular energy metabolism of small intestinal epithelial crypts that could contribute to the increase in crypt proliferation. To test this hypothesis, we utilized primary small intestinal epithelial crypts from C57BL/6J mice to study the effect of glucose on crypt metabolism using a Seahorse XFe96 Extracellular Flux Analyzer for real‐time metabolic measurements. Glycolysis was determined by measuring the extracellular acidification rate (ECAR) in response to glucose and the glycolytic pathway inhibitor 2‐Deoxy‐D‐glucose (2‐DG). Glucose oxidation was determined by measuring oxygen consumption rate (OCR) in response to glucose and the glucose oxidation pathway inhibitor UK‐5099. Glucose‐induced mitochondrial respiration was determined by measuring OCR in response to glucose and modulators of mitochondrial respiration (i.e. ATP synthase inhibitor oligomycin, uncoupler FCCP, complex I inhibitor rotenone, complex III inhibitor antimycin A). The maximum respiration capacity (i.e. the capacity to oxidize any substrates present to meet metabolic challenges) was determined by measuring FCCP‐stimulated OCR. We found that glucose increased ECAR in a concentration dependent manner, and the glycolytic pathway inhibitor 2‐DG reduced glucose‐induced ECAR, indicating that glucose stimulates glycolysis of crypts. However, neither glucose nor the glucose oxidation pathway inhibitor UK‐5099 affected OCR, indicating that glucose oxidation is not active in crypts. Furthermore, glucose increased FCCP‐stimulated OCR, indicating that the presence of glucose is necessary for crypts to utilize substrates for oxidation. These data suggest that high nutrient availability drives metabolism towards utilization of glucose for glycolysis in crypts, and glucose might indirectly induce an increase in the oxidation of other nutrients (e.g. fatty acids, amino acids) in crypts. These metabolic changes may contribute to the nutrient‐induced crypt proliferation.Support or Funding InformationUSDA ILLU‐538‐926