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541 Rotavirus Infection in Patients Is Associated With Altered Trafficking of Apical Membrane Transport Proteins
Incubation of Caco-2 cells with LCA (30 µM) did not significantly alter the mRNA expression of other ion transporters such as PAT-1 or NHE3 indicating the specificity of LCA effects on DRA. Next we investigated the influence of LCA on DRA promoter activity. Caco-2 cells were transiently transfected with full length DRA promoter construct (-1183/+114 bp) and then treated with LCA or CDCA. DRA promoter activity was significantly decreased by LCA but not CDCA. A time-course of LCA treatment (30 µM) demonstrated a decrease in DRA promoter activity as early as 8 h (~50%, P< 0.05) that was sustained for 16 and 24 h (~6070%, p<0.001). These data indicate that LCA (but not other bile acids) decreases DRA expression at the transcriptional level. These effects may underlie the pathophysiology of diarrhea associated with bile acid malabsorption. (Supported by VA & NIDDK)
540 Xenin Induces Anion Secretion via Neural NTS1, NK1 and 5-HT3 Receptors in Rat Duodenum Izumi Kaji, Yasutada Akiba, Jonathan D. Kaunitz Background & Aim: The duodenal mucosa, due to its strategic location, senses luminal nutrients, affects appetite, and regulates duodenal anion secretion, which in turn is important for nutrient absorption and mucosal protection from gastric acid. Xenin-25 is a neurotensin (NT)-related anorexigenic gut hormone, which plasma concentration is increased after meal, and is implicated in anti-diabetic effect. In peripheral tissues, xenin is predominantly expressed in the gastric and duodenal mucosa. Since NT stimulates anion secretion in the hindgut, we hypothesized that xenin stimulates duodenal anion secretion. Methods: A duodenal loop was perfused in an anesthetized rat and luminal bicarbonate secretion was measured with flow-through pH and CO2 electrodes. Electrogenic anion secretion was assessed by measuring short-circuit current (Isc) using Ussing chamber with mucosa-submucosal preparations of rat distal duodenum. Quantitative RT-PCR and immunohistochemistry were used to confirm the expression of xenin and related receptors. Results: Intravenous infusion of xenin-8, a bioactive C-terminal peptide of xenin-25, dose-dependently (1-10 nmol/kg) stimulates duodenal bicarbonate secretion. NTS1 mRNA was highly abundant in the enteric plexus, nodose and dorsal root ganglia, and in the lamina propria rather than in the epithelium; in contrast, NTS2 was abundant in isolated duodenocytes, as well as in neural tissues. The serosal application of xenin-8 and xenin-25 transiently increased Isc, which gradually decreased to baseline in a concentration-dependent manner with an EC50 of 0.3 nM. The NTS1 antagonist SR48692 abolished the response to xenin-8, whereas the NTS2 partial agonist levocabastine had no effect on the basal and xenin-8-evoked Isc, suggesting that NTS1 predominantly mediates xenin-induced duodenal anion secretion. Xenin-8-evoked secretion was inhibited by tetrodotoxin (TTX) or by capsaicin, but not altered by atropine, indicating that a non-cholinergic neural pathway mediates the xenin response. Pretreatment with the NK1 antagonist CP96345 or the 5-HT3 antagonist ondansetron, but not the 5-HT4 antagonist GR113808, reduced the response to xenin-8. NK1 was expressed in extrinsic afferent nerves and submucosal neurons, confirmed by RT-PCR and immunohistochemistry. Xenin-25 was localized in a subset of enteroendocrine cells in the duodenum, but not in the ileum or the colon. Conclusion: Serosal xenin induces duodenal bicarbonate secretion through NTS1 activation on intrinsic and extrinsic afferent nerves, followed by releasing substance P and 5-HT as transmitters. Afferent nerve activation by postprandial, peripherally-released xenin may account for the anorexigenic effect of xenin. Supported by VA Merit Review, NIH R01 DK54221
538 Role of the Intestinal Biotin Uptake System (The Sodium-Dependent Multivitamin Transporter, SMVT) in the Maintenance of Mucosal Integrity Subrata Sabui, Jennifer anne Bohl, Rubina Kapadia, Kyle Cogburn, Nils Lambrecht, Abhisek ghosal, Hamid M. Said Background: The water-soluble, biotin (vitamin B7), is indispensable for normal human health. The vitamin acts as a co-factor for five carboxylases that are critical for fatty acid, glucose and amino acid metabolism. Recently, important roles for biotin in immune function were documented such as activity, generation, maturation and responsiveness of immune cells; it was also found that deficiency of biotin leads to induction in level of pro-inflammatory cytokines. Utilizing a conditional (intestinal-specific) knockout (KO) mouse model, we have recently shown that the SMVT (product of the SLC5A6 gene) is the only uptake system that is involved in intestinal biotin absorption and that its disablement leads to biotin deficiency. During the course of our investigations, however, we observed that all the SMVT KO mice also develop abnormal intestinal pathology and chronic spontaneous inflammation, especially in the cecum. Aim: To investigate the role of SMVT in the maintenance of intestinal mucosal integrity. Methods: Conditional SMVT KO mice and their sex-matched littermates together with a host of physiological and molecular approached were used. Results: Disabling the intestinal SMVT system was associated with: i) a significant increase in gut permeability (measured by FITC-Dextran method); ii) an increase in the expression of "leaky" tight junction (TJ) proteins; and iii) a decrease in expression of "tight" TJ proteins in the cecum. To determine whether these changes are related to the state of biotin deficiency that develops in the SMVT KO animals, we generated a biotin deficiency state in wild-type mice (via feeding a biotin-deficient diet) and examined the effect of this dietary induced-deficiency on different parameters of intestinal integrity; results were compared to their pair-fed controls. Our results showed that dietary biotin deficiency leads to: i) inflammation in the cecal mucosa with an increase in the expression of pro-inflammatory cytokines; ii) an increase in intestinal permeability; and iii) changes in level of expression of TJ proteins similar to those observed with the SMVT KO animals. We also examined the effect of maintaining confluent intestinal epithelial Caco-2 monolayers under chronic biotin deficient condition on permeability and level of expression of TJ proteins, but no change in either parameter was observed compared to cells maintained under biotin sufficient condition. Conclusions: The intestinal SMVT system plays an important role in the maintenance of normal intestinal mucosal integrity. This effect is most likely mediated via its role in securing biotin availability and the known roles biotin plays in normal immunity. Supported by grants from the DVA and the NIH (DK58057 and DK56061).
541 Rotavirus Infection in Patients Is Associated With Altered Trafficking of Apical Membrane Transport Proteins Nicholas W. Baetz, Akshita Gupta, Anirudh Kapoor, Olga Kovbasnjuk, Jerrold Turner, Mark Donowitz, Mary Estes, Nicholas C. Zachos Introduction: Rotavirus (RV) is the leading cause of diarrhea-associated morbidity and mortality in children worldwide. Recently, human small intestinal enteroids have been described as a novel ex vivo intestinal epithelial model to understand RV pathogenesis. To further characterize RV pathophysiology, we tested the hypothesis that RV infection in human small intestinal enteroid monolayers alters the expression and/or localization of ion transporters/channels as well as affects epithelial barrier integrity similar to that observed in biopsies from RV-infected patients. Methods: A) Formalin-fixed paraffin-embedded small intestinal specimens from patients diagnosed with RV diarrhea (confirmed by anti-RV antibody) were analyzed by immunofluorescence confocal microscopy for changes in apical/ basolateral ion/nutrient transporters/channels as well as tight junctional and cytoskeletal proteins and compared to matched control specimens from healthy subjects. B) Proximal small intestinal enteroids generated from biopsies obtained from healthy human subjects were grown as monolayers, differentiated for 5 days, and infected apically with human RV. Changes in protein expression and localization were compared between patient specimens and enteroids. Results: In biopsy specimens from RV-infected patients, antigens for RV and NSP4 were detected in epithelial cells but not in the small intestine of healthy subjects. In RV positive specimens, apical ion/nutrient transporters/channels were downregulated and/or mislocalized compared to small intestinal samples from healthy control subjects. Specifically, NHE2, NHE3, and SGLT-1 were downregulated while CFTR, NHE3, SGLT-1, and the Na+dependent amino acid transporter, SLC6a19, were mislocalized either to intracellular vesicles or the basolateral membrane. In contrast, the basolateral proteins, Na+/K+-ATPase, NKCC1, and b-catenin, as well as tight-junctional proteins, ZO-1 and occludin, were not affected in RV-infected patient samples. The apical cytoskeletal proteins, ezrin and NHERF1, were normally expressed. In RV-infected enteroid monolayers, NHE3, CFTR, SGLT-1 and SLC6a19 were also mislocalized. Conclusions: 1) RV infection results in apical membrane ion/nutrient transporter/channel mislocalization (NHE3, CFTR, SGLT-1, SLC6a19) or down-regulation (NHE2, NHE3, SGLT1) with no change in apical structural, basolateral, or tight junctional proteins. These data suggest RV induces specific pathologic changes in trafficking and/or stability of apical transport proteins. 2) Similar mislocalization of NHE3, CFTR, SGLT-1 and SLC6a19 was observed in RV-infected human small intestinal enteroids suggesting that enteroids accurately reproduce RV-mediated changes at the apical membrane. Human
539 Lithocholic Acid Inhibits the Expression of Cl-/HCO3- Exchanger SLC26A3 in Intestinal Epithelial Cells Shubha Priyamvada, Arivarasu Natarajan Anbazhagan, Anoop Kumar, Megha Singhal, Seema Saksena, Ravinder K. Gill, Waddah A. Alrefai, Pradeep K. Dudeja The majority of bile acids are actively re-absorbed in the distal ileum and only ~ 5% reach the colon in normal conditions. Bile acid malabsorption associated with disorders such as inflammatory bowel disease (IBD) increases the delivery of bile acids to the colon leading to diarrhea. In this regard, SLC26A3 or DRA (Down Regulated in Adenoma) has been shown to be the major anion exchanger mediating Cl- absorption in mammalian intestine. Mutations in DRA gene lead to congenital chloride diarrhea. Also, a reduction in DRA expression in IBD was implicated in the associated diarrhea. However, the role of DRA in bile acid diarrhea is not fully understood. Since bile acids are known to modulate gene expression, we hypothesized that bile acids decrease DRA expression. Intestinal Caco-2 and T-84 cells were used as in vitro models. Apical Cl-/HCO3- exchange activity was measured as a DIDS sensitive 125I uptake. DRA promoter activity was measured by luciferase assays, protein and mRNA expressions were measured by immunoblotting and real time PCR, respectively. Since both the primary (chenodeoxycholic acid, CDCA) and secondary (deoxycholic acid, DCA and lithocholic acid, LCA) bile acids are increased in the intestinal lumen in bile acid malabsorption; we first examined the effects of increasing doses (10-50 µM for 24h) of different bile acids on DRA expression in IECs. LCA treatment for 24h significantly and dose-dependently decreased DRA mRNA and protein expression by a maximum inhibition of ~70% and ~50%, respectively by 30 µM LCA (p<0.05). LCA (30 µM) had no significant effect on cell viability as measured by LDH assay. There was no significant alteration in DRA mRNA expression in response to increasing doses of CDCA and DCA. Further, the effects of LCA on DRA expression were not cell line specific as a similar decrease by LCA occurred in T-84 cells. Treatment with LCA resulted in a dose-dependent inhibition of apical DIDS-sensitive 125I uptake in Caco-2 cells (~52% at 30 µM concentration, P< 0.05).
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studied in vitro), but this did not occur in NHERF3 KO mice. This result was confirmed in Caco-2/Bbe cells with BCECF/fluorometry in which KD of either NHERF3 or NHERF2 prevented STa inhibition of NHE3. In addition, in Caco-2/Bbe cells with NHERF3 KD, the STa inhibition of NHE3 could be rescued by expression of wild-type NHERF3 but not the NHERF3delta4 mutant. We previously showed that the last 4 aa of NHERF3 are involved in NHERF2-NHERF3 hetero-dimerization. c) We and others have shown that there are two plasma membrane complexes associated with GCC-cGMP regulation of NHE3: one involves GCC and the other involves NHE3-NHERF2-cGKII. To test whether one or two signaling complexes are involved, we immunoprecipitated GC-C from mouse small intestine total membrane exposed to STa or PBS for 4 hrs. IP GCC, co-precipitated NHERF2, NHERF3, cGKII, NHE3 and CFTR all of which were increased in amount by STa exposure. Conclusions: 1) STa exposure to small intestine alters a single plasma membrane complex that includes NHERF2, NHERF3, GCC, cGKII, NHE3 and CFTR. With exposure to STa, this represents a single complex that includes two transporters that are phosphorylated by STa/GCC/cGMP (NHE3, CFTR) and two PDZ domain containing scaffolds (NHERF2 and NHERF3). 2) STa alters the activity of NHE3 (reduces) and CFTR (increases). For NHE3, we showed that this regulation is via interactions with a macrocomplex scaffolded by NHERF2-NHERF3 hetero-dimerization that is formed by the last 4 aa of NHERF3 binding to PDZ2 of NHERF2.
enteroid monolayers may serve as a model to understand RV-mediated pathophysiology related to transport physiology and protein trafficking.
544 Antibiotic Altered Microbiota From the Mother Accelerates Development of Colitis in IL-10 Deficient Mice Anjelique Schulfer, Yelina Alvarez, Melody L. Ho, Luc Bijnens, Victoria E. Ruiz, Thomas Battaglia, Laura M. Cox, Arlin Rogers, Martin J. Blaser, R. Balfour Sartor
AGA Abstracts
542 Intrinsic Effects of Reduced NHE3 Activity in Intestinal Epithelial Cells Michael A. Gurney, Daniel Laubitz, Hua Xu, Fayez K. Ghishan, Pawel R. Kiela
Antibiotic use affects the gut microbiota in humans and animal models, and antibioticaltered microbiota (AAM) has been associated with the development of diseases including obesity and atherosclerosis. Epidemiological studies have associated prior antibiotic use with development of inflammatory bowel diseases (IBD), suggesting that an antibiotic-altered microbiota (AAM) may contribute to IBD development. Based on these findings, we asked whether microbiota from antibiotic-exposed mice could exacerbate colitis in genetically predisposed IL-10 deficient (IL-10-/-) recipients. To address this hypothesis, we obtained already characterized cecal microbial populations: AAM that had been isolated from mice treated with low dose antibiotics, previously shown to alter both metabolic and immunologic phenotypes in germ-free recipient mice, and control microbiota (CM) from untreated mice1. These AAM or CM pools were gavaged into pregnant germ-free dams with C57BL/6 wild type (WT) and IL-10 deficient (IL-10-/-) genotypesbackgrounds. For 21 weeks after birth, fecal samples from their pups were collected and IgA and calprotectin were measured at multiple time points. In addition, fecal microbiota 16S rRNA sequencing was performed to assess the transfer efficacy and bacterial population structure in the recipients. Our results show successful transfer of bacteria populations from the pregnant dams to their pups, with congruence in population structures in relation to inoculum (AAM or CM) and to host genotype (WT or IL10-/-). As expected, fecal calprotectin levels were significantly higher in IL-10 -/- compared to WT mice. In WT mice, calprotectin was significantly higher in recipients of AAM compared to CM at week 7. At week 19, IgA concentration was significantly elevated in IL-10 -/- mice compared to WT. However IL-10-/- mice that received AAM had significantly lower IgA levels than those that received CM. Histological staining of colon tissues at sacrifice (week 21) was consistent with colitis development in IL-10 -/-. Notably, the IL-10 -/- mice that received AAM had significantly higher inflammation, hyperplasia, and dysplasia scores than those that received CM. Examination of microbiota in the IL10 -/- mice prior to the histological findings identified specific taxa associated with either acceleration of/or protection from colitis. In summary, our results provide evidence that maternal transfer of an antibiotic-altered microbiota has increased pathogenic effects in offspring predisposed to colitis. Reference: 1. Cox LM, Yamanishi S, Sohn J, Alekseyenko AV, Leung JM, Cho I, et al. (2014) Altering the Intestinal Microbiota during a Critical Developmental Window Has Lasting Metabolic Consequences. Cell 158: 705-721.
Consequences of the inhibition of intestinal NHE3-mediated apical Na+/H+ exchange range from diarrhea to increased susceptibility to mucosal injury or T cell-mediated colitis. Dissection of the cellular mechanisms involved has been challenging due to a number of confounding factors including long-term adaptive responses, microbial dysbiosis, bacterial translocation, and mucosal inflammation. In vitro analysis of NHE3 inhibition was also hindered due to very low or no detectable activity of NHE3 in most colonic cell lines. Methods: Intestinal organoids derived from WT or Slc9a3-/- mice were cultured and analyzed for morphology and ability to propagate. We used SKCO15 cells that express a considerable level of NHE3 mRNA, protein, and specific activity to develop stable lines either with TALEN-mediated inactivating SLC9A3 gene mutation (SLC9A3TALEN), or reduced NHE3 activity via lentiviral shRNA knockdown (SLC9A3KD). Cells were analyzed for morphology, growth, monolayer formation and properties, migration in wound healing, transcriptome (Affymetrix Human Gene 2.0 ST Array) and proteome (Tandem Mass Tag Quantitative Proteomics). Results: Compared to WT mice, primary organoids from Slc9a3-/- were decreased in number, displayed altered morphology and did not survive the first passage. We were not able to culture SLC9A3TALEN cells. SLC9A3KD cells had 75% reduction in NHE3 protein expression and decreased S3226-sensitive initial pH recovery rate. SLC9A3KD cells displayed less-differentiated morphology and attachment deficiency, which could be partly overcome with collagen IV plate coating. SLC9A3KD cells had a significantly higher proliferation rate with doubling time reduced 2.5-fold compared to controls (12.8 vs. 32.3hrs). SLC9A3KD cells demonstrated enhanced migration following wounding and closed the wound to 1% of the initial wound area by 53 or 55 hrs. (2% or 10% serum, respectively), compared to control cells which left 25.3% or 18.0% wound area (2 or 10% serum, respectively) by 72 hrs. Microarray and proteomics analysis indicated significant changes in the gene/protein expression pattern indicative of inhibition of aerobic glycosylation, changes in cell cycle control, cell-matrix and cell-cell interaction, cell migration, epithelium development and differentiation, membrane transport, innate immune response (including IFN g-independent increase in CIITA and MHCII expression), among others. Conclusions: The developed cellular model of reduced NHE3 activity is the first step in understanding the intrinsic defect in the intestinal epithelial cell function as a consequence of NHE3 inhibition during pathogenic or inflammatory challenge. It sheds new light into the roles of uninterrupted epithelial Na+/H+ exchange in normal intestinal epithelium, as well as points to the novel role of NHE3 in modulating colorectal cancer cell behavior and invasiveness.
545 Microbiota From an Active Celiac Donor Modulates Intraepithelial Lymphocyte Numbers and Phenotype in the Mouse Small Intestine Justin McCarville, Marlies Meisel, Heather J. Galipeau, Jennifer Lau, Michael Surette, Yolanda Sanz, Bana Jabri, Elena F. Verdu
543 Epithelial Epidermal Growth Factor Receptor Regulates Colonic Inflammation and Microbiome Composition in Mice Philip E. Dubé, Shivesh Punit, Nandini Girish, Kay Washington, D. Brent Polk
Background: Celiac disease (CeD) is an autoimmune enteropathy triggered by gluten in genetically susceptible individuals harboring the HLA-DQ2 or DQ8 genotype. The disease is characterized by gluten-specific T-cell responses and anti-gluten and anti-tissue transglutaminase 2 autoantibodies. Development of enteropathy, however, requires induction of innate immunity, although the triggers remain unclear. Our aim was to determine whether the microbiota from patients with active CeD induce differential innate immune responses in the small intestine of germ-free (GF) mice, compared to microbiota from healthy controls and investigate underlying mechanisms. Methods: The fecal samples of 7 children with active CeD and 8 healthy age-matched children were utilized in this study. Microbiota was analyzed using 16s rRNA sequencing of the V3 region on a MiSeq platform and analyzed with non-parameteric statistical tests. GF C57BL/6 mice (n=4-5 per donor) were colonized with 100ul of fecal slur by oral gavage from select donors and left for 3 weeks to reach microbial homeostasis. Results: Healthy and active CeD human fecal microbiota demonstrated separate clustering on a Bray-Curtis dissimilarity PCoA plot, with differences characterized by significant decreases in Bacteroidetes (p<0.05) and Proteobacteria (p<0.05) in CeD patients at the phyla level as well as differences within Firmicutes at the genera level. Colonization of GF C57BL/6 mice with microbiota from a HLA-DQ8 heterozygous donor (CeD2), markedly increased intraepithelial lymphocyte (IEL) numbers within small intestinal villi tips compared to colonization with microbiota from the other donors. The phenotype of IELs and inflammatory status of the small intestine was also affected by CeD2 microbiota, with increased proportions of CD3+CD8+abTCR+ IELs (p<0.001), and increased small intestinal IFN-g expression (p<0.05) compared to mice colonized with a selected healthy donor. Sequencing of small intestinal contents in CeD2 recipient mice revealed a higher composition of Proteobacteria (p<0.05), specifically Parasutterella (p<0.05). Further investigation by colonization of MyD88 -/-;Ticam1-/- mice demonstrated that IEL responses to CeD2 colonization were dependent on these signaling adaptors. Conclusions: Our results suggest that there are differences in the composition of the fecal microbiota in CeD patients, which may be a consequence of active disease. However, these results also demonstrate that specific CeD-derived microbiota ecosystems can alter IEL numbers and phenotype in the mouse small intestine through a MyD88/TICAM1 pathway. This mechanism may promote cytotoxic transformation of IELs in some CeD patients, and suggests small intestinal microbiota may be a better biomarker of disease severity than fecal microbiota in these patients.
Epidermal growth factor (EGF) receptor (EGFR) is a therapeutic target for inflammatory bowel disease. EGF enemas were effective to induce remission in a trial of ulcerative colitis patients, and EGFR is protective in mouse colitis models by inducing remission and reducing inflammation. This anti-inflammatory action of EGFR is presumed to be an indirect result of its ability to promote repair. However, reports suggest that intestinal inflammation is separate from barrier dysfunction in a patient with a loss-of-function EGFR mutation or those receiving EGFR inhibitors. Thus, we hypothesized that EGFR directly inhibits epithelial response to bacterial cues, mucosal inflammation, and microbiome composition. Enteroids were established from colonic epithelia of wildtype (WT) mice or mice with a dominantnegative EGFR mutation (EGFRvel), and treated for 3 h ± lipopolysaccharide (LPS) ± EGF. Expression levels of the chemokine CXCL2 were determined by qPCR. Mice expressing a dominant-negative EGFR construct, specifically in the intestinal epithelium (EGFREPI-DN) were administered LPS enemas or saline, as a control; littermates not expressing the transgene were used as controls. Colon tissues were analyzed for histologic scoring. Fecal samples were collected from EGFRvel mice and also from co-housed wildtype (EGFRWT) littermates at 8 wk of age. Bacterial 16S rRNA gene sequences were obtained through Illumina MiSeq and analyzed using QIIME. Microbiome differences among samples were calculated using the weighted or unweighted UniFrac distance measure. EGF significantly inhibited LPS signaling in colonic enteroids. In WT enteroids, LPS induced CXCL2 mRNA expression levels 185-fold (P<0.0001), while EGF co-treatment reduced CXCL2 induction by 52% (P<0.05). EGF alone had no effect. LPS induced CXCL2 expression to a greater extent in EGFRvel enteroids (254-fold), and EGF co-treatment had no effect on this induction, consistent with a specific role for EGFR in the inhibitory effect of EGF. LPS enemas had little effect on wildtype mice, consistent with prior reports. However, EGFREPI-DN mice were highly sensitive to LPS enemas, with lamina propria mononuclear cell and neutrophil infiltration, crypt cell apoptosis and mucosal sloughing. The fecal microbial community structure was different between EGFRWT and EGFRvel littermates with a specific enrichment of Firmicutes genera in EGFRvel feces. EGFR inhibits colonic epithelial responses to LPS (chemokine production and mucosal inflammation), and loss of EGFR induces changes in the colonic microbiome. This reveals a novel anti-inflammatory role for epithelial EGFR through its regulation of epithelial-immune cell crosstalk and host-microbial interactions. Understanding the role of epithelial EGFR in coordinating colonic mucosal inflammation and microbiome composition will lead to next generation therapeutic avenues for IBD.
546 High Sugar Diets Promote an Inflammatory Microbiota and Reduce Gene Expression Related to Intestinal Barrier Function Robert Fedorak, Naomi Hotte, HeeKuk Park, Ammar H. Keshteli, Ruth Ginter, Karen Madsen A breakdown in epithelial barrier function and alterations in gut microbiota are key factors linked with inflammatory bowel disease (IBD). Western diets high in refined sugar have been associated with higher risk of IBD, possibly through diet-induced changes in gut
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540 Xenin Induces Anion Secretion via Neural NTS1, NK1 and 5-HT3 Receptors in Rat Duodenum Izumi Kaji, Yasutada Akiba, Jonathan D. Kaunitz Background & Aim: The duodenal mucosa, due to its strategic location, senses luminal nutrients, affects appetite, and regulates duodenal anion secretion, which in turn is important for nutrient absorption and mucosal protection from gastric acid. Xenin-25 is a neurotensin (NT)-related anorexigenic gut hormone, which plasma concentration is increased after meal, and is implicated in anti-diabetic effect. In peripheral tissues, xenin is predominantly expressed in the gastric and duodenal mucosa. Since NT stimulates anion secretion in the hindgut, we hypothesized that xenin stimulates duodenal anion secretion. Methods: A duodenal loop was perfused in an anesthetized rat and luminal bicarbonate secretion was measured with flow-through pH and CO2 electrodes. Electrogenic anion secretion was assessed by measuring short-circuit current (Isc) using Ussing chamber with mucosa-submucosal preparations of rat distal duodenum. Quantitative RT-PCR and immunohistochemistry were used to confirm the expression of xenin and related receptors. Results: Intravenous infusion of xenin-8, a bioactive C-terminal peptide of xenin-25, dose-dependently (1-10 nmol/kg) stimulates duodenal bicarbonate secretion. NTS1 mRNA was highly abundant in the enteric plexus, nodose and dorsal root ganglia, and in the lamina propria rather than in the epithelium; in contrast, NTS2 was abundant in isolated duodenocytes, as well as in neural tissues. The serosal application of xenin-8 and xenin-25 transiently increased Isc, which gradually decreased to baseline in a concentration-dependent manner with an EC50 of 0.3 nM. The NTS1 antagonist SR48692 abolished the response to xenin-8, whereas the NTS2 partial agonist levocabastine had no effect on the basal and xenin-8-evoked Isc, suggesting that NTS1 predominantly mediates xenin-induced duodenal anion secretion. Xenin-8-evoked secretion was inhibited by tetrodotoxin (TTX) or by capsaicin, but not altered by atropine, indicating that a non-cholinergic neural pathway mediates the xenin response. Pretreatment with the NK1 antagonist CP96345 or the 5-HT3 antagonist ondansetron, but not the 5-HT4 antagonist GR113808, reduced the response to xenin-8. NK1 was expressed in extrinsic afferent nerves and submucosal neurons, confirmed by RT-PCR and immunohistochemistry. Xenin-25 was localized in a subset of enteroendocrine cells in the duodenum, but not in the ileum or the colon. Conclusion: Serosal xenin induces duodenal bicarbonate secretion through NTS1 activation on intrinsic and extrinsic afferent nerves, followed by releasing substance P and 5-HT as transmitters. Afferent nerve activation by postprandial, peripherally-released xenin may account for the anorexigenic effect of xenin. Supported by VA Merit Review, NIH R01 DK54221
538 Role of the Intestinal Biotin Uptake System (The Sodium-Dependent Multivitamin Transporter, SMVT) in the Maintenance of Mucosal Integrity Subrata Sabui, Jennifer anne Bohl, Rubina Kapadia, Kyle Cogburn, Nils Lambrecht, Abhisek ghosal, Hamid M. Said Background: The water-soluble, biotin (vitamin B7), is indispensable for normal human health. The vitamin acts as a co-factor for five carboxylases that are critical for fatty acid, glucose and amino acid metabolism. Recently, important roles for biotin in immune function were documented such as activity, generation, maturation and responsiveness of immune cells; it was also found that deficiency of biotin leads to induction in level of pro-inflammatory cytokines. Utilizing a conditional (intestinal-specific) knockout (KO) mouse model, we have recently shown that the SMVT (product of the SLC5A6 gene) is the only uptake system that is involved in intestinal biotin absorption and that its disablement leads to biotin deficiency. During the course of our investigations, however, we observed that all the SMVT KO mice also develop abnormal intestinal pathology and chronic spontaneous inflammation, especially in the cecum. Aim: To investigate the role of SMVT in the maintenance of intestinal mucosal integrity. Methods: Conditional SMVT KO mice and their sex-matched littermates together with a host of physiological and molecular approached were used. Results: Disabling the intestinal SMVT system was associated with: i) a significant increase in gut permeability (measured by FITC-Dextran method); ii) an increase in the expression of "leaky" tight junction (TJ) proteins; and iii) a decrease in expression of "tight" TJ proteins in the cecum. To determine whether these changes are related to the state of biotin deficiency that develops in the SMVT KO animals, we generated a biotin deficiency state in wild-type mice (via feeding a biotin-deficient diet) and examined the effect of this dietary induced-deficiency on different parameters of intestinal integrity; results were compared to their pair-fed controls. Our results showed that dietary biotin deficiency leads to: i) inflammation in the cecal mucosa with an increase in the expression of pro-inflammatory cytokines; ii) an increase in intestinal permeability; and iii) changes in level of expression of TJ proteins similar to those observed with the SMVT KO animals. We also examined the effect of maintaining confluent intestinal epithelial Caco-2 monolayers under chronic biotin deficient condition on permeability and level of expression of TJ proteins, but no change in either parameter was observed compared to cells maintained under biotin sufficient condition. Conclusions: The intestinal SMVT system plays an important role in the maintenance of normal intestinal mucosal integrity. This effect is most likely mediated via its role in securing biotin availability and the known roles biotin plays in normal immunity. Supported by grants from the DVA and the NIH (DK58057 and DK56061).
541 Rotavirus Infection in Patients Is Associated With Altered Trafficking of Apical Membrane Transport Proteins Nicholas W. Baetz, Akshita Gupta, Anirudh Kapoor, Olga Kovbasnjuk, Jerrold Turner, Mark Donowitz, Mary Estes, Nicholas C. Zachos Introduction: Rotavirus (RV) is the leading cause of diarrhea-associated morbidity and mortality in children worldwide. Recently, human small intestinal enteroids have been described as a novel ex vivo intestinal epithelial model to understand RV pathogenesis. To further characterize RV pathophysiology, we tested the hypothesis that RV infection in human small intestinal enteroid monolayers alters the expression and/or localization of ion transporters/channels as well as affects epithelial barrier integrity similar to that observed in biopsies from RV-infected patients. Methods: A) Formalin-fixed paraffin-embedded small intestinal specimens from patients diagnosed with RV diarrhea (confirmed by anti-RV antibody) were analyzed by immunofluorescence confocal microscopy for changes in apical/ basolateral ion/nutrient transporters/channels as well as tight junctional and cytoskeletal proteins and compared to matched control specimens from healthy subjects. B) Proximal small intestinal enteroids generated from biopsies obtained from healthy human subjects were grown as monolayers, differentiated for 5 days, and infected apically with human RV. Changes in protein expression and localization were compared between patient specimens and enteroids. Results: In biopsy specimens from RV-infected patients, antigens for RV and NSP4 were detected in epithelial cells but not in the small intestine of healthy subjects. In RV positive specimens, apical ion/nutrient transporters/channels were downregulated and/or mislocalized compared to small intestinal samples from healthy control subjects. Specifically, NHE2, NHE3, and SGLT-1 were downregulated while CFTR, NHE3, SGLT-1, and the Na+dependent amino acid transporter, SLC6a19, were mislocalized either to intracellular vesicles or the basolateral membrane. In contrast, the basolateral proteins, Na+/K+-ATPase, NKCC1, and b-catenin, as well as tight-junctional proteins, ZO-1 and occludin, were not affected in RV-infected patient samples. The apical cytoskeletal proteins, ezrin and NHERF1, were normally expressed. In RV-infected enteroid monolayers, NHE3, CFTR, SGLT-1 and SLC6a19 were also mislocalized. Conclusions: 1) RV infection results in apical membrane ion/nutrient transporter/channel mislocalization (NHE3, CFTR, SGLT-1, SLC6a19) or down-regulation (NHE2, NHE3, SGLT1) with no change in apical structural, basolateral, or tight junctional proteins. These data suggest RV induces specific pathologic changes in trafficking and/or stability of apical transport proteins. 2) Similar mislocalization of NHE3, CFTR, SGLT-1 and SLC6a19 was observed in RV-infected human small intestinal enteroids suggesting that enteroids accurately reproduce RV-mediated changes at the apical membrane. Human
539 Lithocholic Acid Inhibits the Expression of Cl-/HCO3- Exchanger SLC26A3 in Intestinal Epithelial Cells Shubha Priyamvada, Arivarasu Natarajan Anbazhagan, Anoop Kumar, Megha Singhal, Seema Saksena, Ravinder K. Gill, Waddah A. Alrefai, Pradeep K. Dudeja The majority of bile acids are actively re-absorbed in the distal ileum and only ~ 5% reach the colon in normal conditions. Bile acid malabsorption associated with disorders such as inflammatory bowel disease (IBD) increases the delivery of bile acids to the colon leading to diarrhea. In this regard, SLC26A3 or DRA (Down Regulated in Adenoma) has been shown to be the major anion exchanger mediating Cl- absorption in mammalian intestine. Mutations in DRA gene lead to congenital chloride diarrhea. Also, a reduction in DRA expression in IBD was implicated in the associated diarrhea. However, the role of DRA in bile acid diarrhea is not fully understood. Since bile acids are known to modulate gene expression, we hypothesized that bile acids decrease DRA expression. Intestinal Caco-2 and T-84 cells were used as in vitro models. Apical Cl-/HCO3- exchange activity was measured as a DIDS sensitive 125I uptake. DRA promoter activity was measured by luciferase assays, protein and mRNA expressions were measured by immunoblotting and real time PCR, respectively. Since both the primary (chenodeoxycholic acid, CDCA) and secondary (deoxycholic acid, DCA and lithocholic acid, LCA) bile acids are increased in the intestinal lumen in bile acid malabsorption; we first examined the effects of increasing doses (10-50 µM for 24h) of different bile acids on DRA expression in IECs. LCA treatment for 24h significantly and dose-dependently decreased DRA mRNA and protein expression by a maximum inhibition of ~70% and ~50%, respectively by 30 µM LCA (p<0.05). LCA (30 µM) had no significant effect on cell viability as measured by LDH assay. There was no significant alteration in DRA mRNA expression in response to increasing doses of CDCA and DCA. Further, the effects of LCA on DRA expression were not cell line specific as a similar decrease by LCA occurred in T-84 cells. Treatment with LCA resulted in a dose-dependent inhibition of apical DIDS-sensitive 125I uptake in Caco-2 cells (~52% at 30 µM concentration, P< 0.05).
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studied in vitro), but this did not occur in NHERF3 KO mice. This result was confirmed in Caco-2/Bbe cells with BCECF/fluorometry in which KD of either NHERF3 or NHERF2 prevented STa inhibition of NHE3. In addition, in Caco-2/Bbe cells with NHERF3 KD, the STa inhibition of NHE3 could be rescued by expression of wild-type NHERF3 but not the NHERF3delta4 mutant. We previously showed that the last 4 aa of NHERF3 are involved in NHERF2-NHERF3 hetero-dimerization. c) We and others have shown that there are two plasma membrane complexes associated with GCC-cGMP regulation of NHE3: one involves GCC and the other involves NHE3-NHERF2-cGKII. To test whether one or two signaling complexes are involved, we immunoprecipitated GC-C from mouse small intestine total membrane exposed to STa or PBS for 4 hrs. IP GCC, co-precipitated NHERF2, NHERF3, cGKII, NHE3 and CFTR all of which were increased in amount by STa exposure. Conclusions: 1) STa exposure to small intestine alters a single plasma membrane complex that includes NHERF2, NHERF3, GCC, cGKII, NHE3 and CFTR. With exposure to STa, this represents a single complex that includes two transporters that are phosphorylated by STa/GCC/cGMP (NHE3, CFTR) and two PDZ domain containing scaffolds (NHERF2 and NHERF3). 2) STa alters the activity of NHE3 (reduces) and CFTR (increases). For NHE3, we showed that this regulation is via interactions with a macrocomplex scaffolded by NHERF2-NHERF3 hetero-dimerization that is formed by the last 4 aa of NHERF3 binding to PDZ2 of NHERF2.
enteroid monolayers may serve as a model to understand RV-mediated pathophysiology related to transport physiology and protein trafficking.
544 Antibiotic Altered Microbiota From the Mother Accelerates Development of Colitis in IL-10 Deficient Mice Anjelique Schulfer, Yelina Alvarez, Melody L. Ho, Luc Bijnens, Victoria E. Ruiz, Thomas Battaglia, Laura M. Cox, Arlin Rogers, Martin J. Blaser, R. Balfour Sartor
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542 Intrinsic Effects of Reduced NHE3 Activity in Intestinal Epithelial Cells Michael A. Gurney, Daniel Laubitz, Hua Xu, Fayez K. Ghishan, Pawel R. Kiela
Antibiotic use affects the gut microbiota in humans and animal models, and antibioticaltered microbiota (AAM) has been associated with the development of diseases including obesity and atherosclerosis. Epidemiological studies have associated prior antibiotic use with development of inflammatory bowel diseases (IBD), suggesting that an antibiotic-altered microbiota (AAM) may contribute to IBD development. Based on these findings, we asked whether microbiota from antibiotic-exposed mice could exacerbate colitis in genetically predisposed IL-10 deficient (IL-10-/-) recipients. To address this hypothesis, we obtained already characterized cecal microbial populations: AAM that had been isolated from mice treated with low dose antibiotics, previously shown to alter both metabolic and immunologic phenotypes in germ-free recipient mice, and control microbiota (CM) from untreated mice1. These AAM or CM pools were gavaged into pregnant germ-free dams with C57BL/6 wild type (WT) and IL-10 deficient (IL-10-/-) genotypesbackgrounds. For 21 weeks after birth, fecal samples from their pups were collected and IgA and calprotectin were measured at multiple time points. In addition, fecal microbiota 16S rRNA sequencing was performed to assess the transfer efficacy and bacterial population structure in the recipients. Our results show successful transfer of bacteria populations from the pregnant dams to their pups, with congruence in population structures in relation to inoculum (AAM or CM) and to host genotype (WT or IL10-/-). As expected, fecal calprotectin levels were significantly higher in IL-10 -/- compared to WT mice. In WT mice, calprotectin was significantly higher in recipients of AAM compared to CM at week 7. At week 19, IgA concentration was significantly elevated in IL-10 -/- mice compared to WT. However IL-10-/- mice that received AAM had significantly lower IgA levels than those that received CM. Histological staining of colon tissues at sacrifice (week 21) was consistent with colitis development in IL-10 -/-. Notably, the IL-10 -/- mice that received AAM had significantly higher inflammation, hyperplasia, and dysplasia scores than those that received CM. Examination of microbiota in the IL10 -/- mice prior to the histological findings identified specific taxa associated with either acceleration of/or protection from colitis. In summary, our results provide evidence that maternal transfer of an antibiotic-altered microbiota has increased pathogenic effects in offspring predisposed to colitis. Reference: 1. Cox LM, Yamanishi S, Sohn J, Alekseyenko AV, Leung JM, Cho I, et al. (2014) Altering the Intestinal Microbiota during a Critical Developmental Window Has Lasting Metabolic Consequences. Cell 158: 705-721.
Consequences of the inhibition of intestinal NHE3-mediated apical Na+/H+ exchange range from diarrhea to increased susceptibility to mucosal injury or T cell-mediated colitis. Dissection of the cellular mechanisms involved has been challenging due to a number of confounding factors including long-term adaptive responses, microbial dysbiosis, bacterial translocation, and mucosal inflammation. In vitro analysis of NHE3 inhibition was also hindered due to very low or no detectable activity of NHE3 in most colonic cell lines. Methods: Intestinal organoids derived from WT or Slc9a3-/- mice were cultured and analyzed for morphology and ability to propagate. We used SKCO15 cells that express a considerable level of NHE3 mRNA, protein, and specific activity to develop stable lines either with TALEN-mediated inactivating SLC9A3 gene mutation (SLC9A3TALEN), or reduced NHE3 activity via lentiviral shRNA knockdown (SLC9A3KD). Cells were analyzed for morphology, growth, monolayer formation and properties, migration in wound healing, transcriptome (Affymetrix Human Gene 2.0 ST Array) and proteome (Tandem Mass Tag Quantitative Proteomics). Results: Compared to WT mice, primary organoids from Slc9a3-/- were decreased in number, displayed altered morphology and did not survive the first passage. We were not able to culture SLC9A3TALEN cells. SLC9A3KD cells had 75% reduction in NHE3 protein expression and decreased S3226-sensitive initial pH recovery rate. SLC9A3KD cells displayed less-differentiated morphology and attachment deficiency, which could be partly overcome with collagen IV plate coating. SLC9A3KD cells had a significantly higher proliferation rate with doubling time reduced 2.5-fold compared to controls (12.8 vs. 32.3hrs). SLC9A3KD cells demonstrated enhanced migration following wounding and closed the wound to 1% of the initial wound area by 53 or 55 hrs. (2% or 10% serum, respectively), compared to control cells which left 25.3% or 18.0% wound area (2 or 10% serum, respectively) by 72 hrs. Microarray and proteomics analysis indicated significant changes in the gene/protein expression pattern indicative of inhibition of aerobic glycosylation, changes in cell cycle control, cell-matrix and cell-cell interaction, cell migration, epithelium development and differentiation, membrane transport, innate immune response (including IFN g-independent increase in CIITA and MHCII expression), among others. Conclusions: The developed cellular model of reduced NHE3 activity is the first step in understanding the intrinsic defect in the intestinal epithelial cell function as a consequence of NHE3 inhibition during pathogenic or inflammatory challenge. It sheds new light into the roles of uninterrupted epithelial Na+/H+ exchange in normal intestinal epithelium, as well as points to the novel role of NHE3 in modulating colorectal cancer cell behavior and invasiveness.
545 Microbiota From an Active Celiac Donor Modulates Intraepithelial Lymphocyte Numbers and Phenotype in the Mouse Small Intestine Justin McCarville, Marlies Meisel, Heather J. Galipeau, Jennifer Lau, Michael Surette, Yolanda Sanz, Bana Jabri, Elena F. Verdu
543 Epithelial Epidermal Growth Factor Receptor Regulates Colonic Inflammation and Microbiome Composition in Mice Philip E. Dubé, Shivesh Punit, Nandini Girish, Kay Washington, D. Brent Polk
Background: Celiac disease (CeD) is an autoimmune enteropathy triggered by gluten in genetically susceptible individuals harboring the HLA-DQ2 or DQ8 genotype. The disease is characterized by gluten-specific T-cell responses and anti-gluten and anti-tissue transglutaminase 2 autoantibodies. Development of enteropathy, however, requires induction of innate immunity, although the triggers remain unclear. Our aim was to determine whether the microbiota from patients with active CeD induce differential innate immune responses in the small intestine of germ-free (GF) mice, compared to microbiota from healthy controls and investigate underlying mechanisms. Methods: The fecal samples of 7 children with active CeD and 8 healthy age-matched children were utilized in this study. Microbiota was analyzed using 16s rRNA sequencing of the V3 region on a MiSeq platform and analyzed with non-parameteric statistical tests. GF C57BL/6 mice (n=4-5 per donor) were colonized with 100ul of fecal slur by oral gavage from select donors and left for 3 weeks to reach microbial homeostasis. Results: Healthy and active CeD human fecal microbiota demonstrated separate clustering on a Bray-Curtis dissimilarity PCoA plot, with differences characterized by significant decreases in Bacteroidetes (p<0.05) and Proteobacteria (p<0.05) in CeD patients at the phyla level as well as differences within Firmicutes at the genera level. Colonization of GF C57BL/6 mice with microbiota from a HLA-DQ8 heterozygous donor (CeD2), markedly increased intraepithelial lymphocyte (IEL) numbers within small intestinal villi tips compared to colonization with microbiota from the other donors. The phenotype of IELs and inflammatory status of the small intestine was also affected by CeD2 microbiota, with increased proportions of CD3+CD8+abTCR+ IELs (p<0.001), and increased small intestinal IFN-g expression (p<0.05) compared to mice colonized with a selected healthy donor. Sequencing of small intestinal contents in CeD2 recipient mice revealed a higher composition of Proteobacteria (p<0.05), specifically Parasutterella (p<0.05). Further investigation by colonization of MyD88 -/-;Ticam1-/- mice demonstrated that IEL responses to CeD2 colonization were dependent on these signaling adaptors. Conclusions: Our results suggest that there are differences in the composition of the fecal microbiota in CeD patients, which may be a consequence of active disease. However, these results also demonstrate that specific CeD-derived microbiota ecosystems can alter IEL numbers and phenotype in the mouse small intestine through a MyD88/TICAM1 pathway. This mechanism may promote cytotoxic transformation of IELs in some CeD patients, and suggests small intestinal microbiota may be a better biomarker of disease severity than fecal microbiota in these patients.
Epidermal growth factor (EGF) receptor (EGFR) is a therapeutic target for inflammatory bowel disease. EGF enemas were effective to induce remission in a trial of ulcerative colitis patients, and EGFR is protective in mouse colitis models by inducing remission and reducing inflammation. This anti-inflammatory action of EGFR is presumed to be an indirect result of its ability to promote repair. However, reports suggest that intestinal inflammation is separate from barrier dysfunction in a patient with a loss-of-function EGFR mutation or those receiving EGFR inhibitors. Thus, we hypothesized that EGFR directly inhibits epithelial response to bacterial cues, mucosal inflammation, and microbiome composition. Enteroids were established from colonic epithelia of wildtype (WT) mice or mice with a dominantnegative EGFR mutation (EGFRvel), and treated for 3 h ± lipopolysaccharide (LPS) ± EGF. Expression levels of the chemokine CXCL2 were determined by qPCR. Mice expressing a dominant-negative EGFR construct, specifically in the intestinal epithelium (EGFREPI-DN) were administered LPS enemas or saline, as a control; littermates not expressing the transgene were used as controls. Colon tissues were analyzed for histologic scoring. Fecal samples were collected from EGFRvel mice and also from co-housed wildtype (EGFRWT) littermates at 8 wk of age. Bacterial 16S rRNA gene sequences were obtained through Illumina MiSeq and analyzed using QIIME. Microbiome differences among samples were calculated using the weighted or unweighted UniFrac distance measure. EGF significantly inhibited LPS signaling in colonic enteroids. In WT enteroids, LPS induced CXCL2 mRNA expression levels 185-fold (P<0.0001), while EGF co-treatment reduced CXCL2 induction by 52% (P<0.05). EGF alone had no effect. LPS induced CXCL2 expression to a greater extent in EGFRvel enteroids (254-fold), and EGF co-treatment had no effect on this induction, consistent with a specific role for EGFR in the inhibitory effect of EGF. LPS enemas had little effect on wildtype mice, consistent with prior reports. However, EGFREPI-DN mice were highly sensitive to LPS enemas, with lamina propria mononuclear cell and neutrophil infiltration, crypt cell apoptosis and mucosal sloughing. The fecal microbial community structure was different between EGFRWT and EGFRvel littermates with a specific enrichment of Firmicutes genera in EGFRvel feces. EGFR inhibits colonic epithelial responses to LPS (chemokine production and mucosal inflammation), and loss of EGFR induces changes in the colonic microbiome. This reveals a novel anti-inflammatory role for epithelial EGFR through its regulation of epithelial-immune cell crosstalk and host-microbial interactions. Understanding the role of epithelial EGFR in coordinating colonic mucosal inflammation and microbiome composition will lead to next generation therapeutic avenues for IBD.
546 High Sugar Diets Promote an Inflammatory Microbiota and Reduce Gene Expression Related to Intestinal Barrier Function Robert Fedorak, Naomi Hotte, HeeKuk Park, Ammar H. Keshteli, Ruth Ginter, Karen Madsen A breakdown in epithelial barrier function and alterations in gut microbiota are key factors linked with inflammatory bowel disease (IBD). Western diets high in refined sugar have been associated with higher risk of IBD, possibly through diet-induced changes in gut
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