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Su1867 Characterization of Epithelial Barrier Function in Intestinal Graft-Versus-Host Disease
increase; p<0.05) and a parallel reduction in TER. Histological evaluation of the small intestine and colon revealed marked intestinal hemorrhage in CRF2-/- mice following induction of PSA. The increase in intestinal hemorrhage coincided with a significant reduction in the cardiac blood volume in CRF2 deficient mice (68% and 49% reduction compared to WT mice at 30 and 120 min post DNP treatment). To specifically examine the role of mast cell specific CRF2 in PSA, we induced PSA in mast cell deficient mice (Kit W-sh/W-sh) that were reconstituted with either WT or CRF2-/- bone marrow derived mast cells (BMMCs). Compared with mast cell deficient mice reconstituted with WT BMMCs, mice repleted with CRF2-/BMMCs responded with higher histamine release and greater reduction in body temperature following PSA induction. Together, these results indicate that mast cell CRF2 plays a novel role in regulating IgE-mediated mast cell degranulation, intestinal injury, and anaphylactic symptoms. Su1867 Characterization of Epithelial Barrier Function in Intestinal Graft-Versus-Host Disease Hanno Troeger, Nina Hering, Kathrin Rieger, Britta Siegmund, Joerg D. Schulzke Background & Aims: In acute intestinal graft-versus-host disease (A-GVHD) epithelial barrier function is important both in perpetuating the mucosal inflammation as well as for the pathomechanism of diarrhea. This study aimed to characterize the intestinal barrier function in human A-GVHD using an established biopsy ex vivo model. Methods: Sigmoid biopsies from 14 patients with A-GVHD and 15 controls were obtained endoscopically. A-GVHD was diagnosed by clinical judgment in combination with histological analysis. Short circuit current (ISC) and permeabilities to mannitol and horse-radish-peroxidase (HRP) were measured in miniaturized Ussing chambers. Resistances of the epithelial layer and of subepithelial layers were determined by impedance spectroscopy. Additional biopsy specimens were used for expression and localization of tight junction proteins by western blotting and immunohistology using confocal microscopy. Results: Epithelial resistance was decreased from 36.0± 3.4 Vcm2 in controls to 19.0±3.4 Vcm2 in A-GVHD (p<0.01). In contrast, ISC was not significantly altered. Mannitol flux was 240±21 nmol h-1cm-2 in controls and increased to 536±104 nmol h-1cm-2 in A-GVHD specimens. Likewise, HRP-flux was significantly increased in A-GVHD. Caspase-3 -stained immunohistological sections of colonic biopsies showed increased epithelial apoptosis compared to controls. Western Blot analysis revealed an increased expression of claudin-2 only in severe A-GVHD. Conclusions: For the first time, this study showed direct evidence for a barrier dysfunction in human A-GVHD with respect to ions, small tracers (mannitol) and also for high molecular weight markers (HRP). Barrier defects were due to the increased apoptotic rate and expression of the poreforming claudin-2 in later stages. These findings support the general concept of a barrier dysfunction in the pathogenesis of A-GVHD as well as a role for leak flux as a pathomechanism of diarrhea in this disease.
Su1865 Mechanism of Autophagy Regulation of Intestinal Epithelial TJ Barrier: Role of Claudin-2 in TJ Barrier Enhancement Prashant K. Nighot, Shuhong Guo, Manmeet Rawat, Chien-An A. Hu, Thomas Y. Ma Background: Autophagy is a cellular degradation pathway and is considered to be a cell survival mechanism. Defects in autophagy are implicated in many pathological processes including Inflammatory Bowel Disease. Among the innate defense mechanisms of intestinal mucosa, defective tight junction (TJ) barrier has been postulated as a key pathogenic factor in causation and progression of Inflammatory Bowel Disease. The cross talk between autophagy and TJ barrier has not yet been studied. Aim: The aim of this study was to investigate the role of autophagy in intestinal TJ barrier function. Methods: We used pharmacologic and genetic autophagy modulation in a cell culture model of filter grown human intestinal Caco-2 monolayers to study the effect of autophagy on intestinal epithelial TJ barrier. Results: Induction of nutrient starvation-induced autophagy by incubation of Caco-2 monolayers in serum and amino acids free media increased transepithelial resistance (TER) up to 200% over 96 hours. Induction of autophagy was confirmed by immunoblotting of microtubuleassociated protein light chain 3 (LC3) where the ratio of LC3-II to LC3-I was found to be increased over the experimental period of 96 hours. Confocal immunofluorescence revealed typical punctate cytoplasmic fluorescence for LC3 after starvation. The dilution potential as well as ratio of sodium/chloride permeability was significantly decreased in starved cells compared to control full-fed cells. The paracellular permeability of urea (molecular radius 2.9 Å), but not mannitol (4.1 Å), inulin (15 Å), and 10-kDa dextran (23 Å) was significantly reduced in starved cells. Treatment of nutrient fed Caco-2 monolayers with pharmacologic autophagy inducers rapamycin and PP242 also caused similar increase in TER comparable to that of control starved Caco-2 manolayers, and reduced paracellular urea flux. On the other hand, autophagy inhibitors bafilomycin A1, chloroquine, and wortmannin attenuated starvation-induced increase in TER. Also, siRNA silencing of autophagy-related protein ATG16L1 and ATG7, but not transfection with non-targeted siRNA, significantly inhibited starvation-induced increase in TER and reduction in paracellular urea flux. Immunoblotting for TJ proteins showed a remarkable decrease in the level of pore forming claudin-2 and an increase in level of occludin after starvation. Starvation did not affect claudin-1,-3,-8, and ZO-1 protein levels. In confocal immunofluorescence, reduced membrane immunostaining and increased cytoplasmic colocalization with lysosome marker LAMP-2 was seen for claudin-2 but not occludin, indicating specific degradation of claudin-2 during induction of autophagy. Conclusion: Overall, our data show that autophagy reduces paracellular TJ permeability of ions and small molecules by down-regulation TJ protein claudin-2.
Su1868 Constant Stimulation of Epithelial Cell Death in the Steady-State Gut Is Controlled via CFLIP Nadine Wittkopf, Claudia Günther, Eva Martini, You-Wen He, Marcus Schuchmann, Markus F. Neurath, Christoph Becker Introduction: The gut is composed of several cell types including intestinal epithelial cells (IECs) and immune cells. The complex interaction between immune cells and intestinal epithelial cells is of utmost importance for gut homeostasis and has been demonstrated to be involved in diseases of the gut such as inflammatory bowel disease, intestinal infections, and colorectal cancer. In the steady-state gut and in the course of gut diseases, intestinal epithelial cells not only signal information about the gut lumen to the subjacent immune cells, most importantly, they respond to mediators produced by immune cells which thereby shape the behavior of IECs in an appropriate way. Methods: Mice with an inducible deletion of cFlip (cellular FLICE-like inhibitory protein) in IECs (cFlipiΔIEC) were generated by crossbreeding cFlipfl mice to Vil-CreERT2 mice. The influence of external cell death triggers in the gut on intestinal epithelial cell death was investigated by organoid culture, endoscopy, immunofluorescence stainings, western blot and qPCR. Results: Deletion of the caspase-8 inhibitor cFlip in intestinal epithelial cells in vivo caused excessive death of IECs in both villi and crypts along with breakdown of intestinal epithelial barrier integrity, inflammation, loss of tissue architecture and ultimately led to death of mice. In striking contrast, deletion of cFlip in crypt epithelial cells in organoid culture in vitro did not result in intestinal epithelial cell death indicating that constant cell death triggers were present in the steadystate gut of cFlipiΔIEC mice but not in organoid culture. Accordingly, death of cFlip deficient organoids was achieved after external addition of cell death receptor ligands such as TNFα (tumor necrosis factor alpha) or CD95L (CD95 ligand). Histological analysis of cFlip deficient organoids demonstrated them to stain highly positive for activated caspase-3 and activated caspase-8 and therefore to die via apoptosis. Interestingly, the cell death mediators present in the steady-state gut and inducing apoptosis in cFlip deficient IECs seemed to be independent from age of mice and probably from the gut flora: cFlip deficient mice died in utero and mice at age 0 weeks, 2 weeks, 5 weeks, and 15 weeks died at similar time points after inducing cflip deficiency and showed comparable IEC death and loss of tissue architecture. Conclusion: Our data suggest the constant presence of cell death triggers in the uninflamed steady-state gut. The activation of intestinal epithelial cell death needs to be tightly regulated to prevent from severe gut dysfunction. cFlip turned out to be a central regulator of IEC behavior responding to the cell death triggers constantly present in the gut.
Su1866 Mast Cell Corticotropin Releasing Factor Receptor 2 Regulates IgE-Mediated Mast Cell Degranulation, Anaphylaxis, and Intestinal Permeability Susan D'Costa, Laura Edwards, Adam J. Moeser The corticotropin releasing factor (CRF) system composed of CRF, related urocortin ligands and receptors CRF1 and CRF2 is the primary regulator of the stress response and a key player in stress-related GI diseases. While the CRF system has been studied extensively in the nervous system, its role in regulating the immune system remains poorly defined. The specific objective of this study was to determine the role of CRF2 in the regulation of immunological responses and intestinal injury induced by IgE-mediated passive systemic anaphylaxis (PSA). To induce PSA, wildtype and CRF2-/- mice were sensitized with antidinitrophenyl (DNP)-IgE overnight followed by administration of DNP-HSA antigen. No significant genotypic differences were found in body temperature and histamine levels prior to antigen injection. Wildtype (WT) and CRF2-/- mice exhibited a similar degree of hypothermia following PSA. However, compared with WT mice, CRF2-/- mice exhibited markedly increased mast cell degranulation indicated by higher serum histamine levels (3.9fold, p<0.05) and histological evaluation. To evaluate the effect of anaphylactic stress on the gut, the jejunum and colon were harvested from mice 120 minutes post-PSA and mounted on the Ussing Chamber for measurements of Transepithelial Electrical Resistance (TER) and 4 kDa-FITC dextran (FD4) flux. Compared to wildtype mice, CRF2-/- mice had increased FD4 permeability across jejunal (41% increase; p<0.05) and colonic mucosa (160%
S-489
AGA Abstracts
AGA Abstracts
investigate the relationship between p53 status of cancer cells and activation of fibroblasts was estimated pathologically using human colorectal cancer tissues. We used HCT116, a cultured cell line derived from human colon cancer tissue: p53-wild type HCT116p53+/+, p53-knockout HCT116p53-/-. Furthermore, we established p53-suppressed cell line using p53 shRNA (HCT116sh p53) compared with HCT116sh control. To elucidate the effect of fibroblast existence on tumor growth, in in vivo study, HCT116 p53+/+ , HCT116 p53-/-, HCT116sh control and HCT116sh p53 were implanted subcutaneously into nude mice, with or without WI-38, a human fibroblast cell line, and the volume of implanted subcutaneous tumors were determined. In in vitro study, the gene expression signature of HCT116 cells co-cultured directly or through transwell filters with or without WI-38 was determined. Immunohistochemistry study of human colon cancer tissues revealed the significant positive relationship among p53 dysfunction, fibroblast agglomeration, and micro vessel density. Xenograft tumors of p53-dysfunctional cells (HCT116p53-/- or HCT116 sh p53) implanted with WI-38 cells grew up larger than tumors of p53-functional cells (HCT116p53+/ + or HCT116sh control) with or without WI-38 cells, and p53 dysfunctional cells without WI38 cells. In in vitro, no significant increase in proliferation of p53-normal or p53-functionaldeficient HCT116 cells co-cultured with WI-38, directly or through transwell, was detected compared without WI-38. WI-38 co-cultured with p53 dysfunctional cells secreted more VEGF than the one with p53 normal cells, indicating that p53 dysfunctional cancer cells induced alteration of angiogenic characteristics from fibroblasts to CAFs. VEGF deletion in WI-38, not cancer cells, reduced xenograft tumor size of p53 dysfunctional cells implanted with WI-38. These results demonstrated that VEGF produced by fibroblasts, not epithelial cancer cells, has a crucial role in p53-functional-deficient tumor growth. The present study indicates that p53-incompetent tumor cells educate fibroblasts to secrete VEGF, leading to tumor progression. This is the novel insight in that gene alteration of epithelial cancer cells triggers the transformation of tumor microenvironment.
Su1865 Mechanism of Autophagy Regulation of Intestinal Epithelial TJ Barrier: Role of Claudin-2 in TJ Barrier Enhancement Prashant K. Nighot, Shuhong Guo, Manmeet Rawat, Chien-An A. Hu, Thomas Y. Ma Background: Autophagy is a cellular degradation pathway and is considered to be a cell survival mechanism. Defects in autophagy are implicated in many pathological processes including Inflammatory Bowel Disease. Among the innate defense mechanisms of intestinal mucosa, defective tight junction (TJ) barrier has been postulated as a key pathogenic factor in causation and progression of Inflammatory Bowel Disease. The cross talk between autophagy and TJ barrier has not yet been studied. Aim: The aim of this study was to investigate the role of autophagy in intestinal TJ barrier function. Methods: We used pharmacologic and genetic autophagy modulation in a cell culture model of filter grown human intestinal Caco-2 monolayers to study the effect of autophagy on intestinal epithelial TJ barrier. Results: Induction of nutrient starvation-induced autophagy by incubation of Caco-2 monolayers in serum and amino acids free media increased transepithelial resistance (TER) up to 200% over 96 hours. Induction of autophagy was confirmed by immunoblotting of microtubuleassociated protein light chain 3 (LC3) where the ratio of LC3-II to LC3-I was found to be increased over the experimental period of 96 hours. Confocal immunofluorescence revealed typical punctate cytoplasmic fluorescence for LC3 after starvation. The dilution potential as well as ratio of sodium/chloride permeability was significantly decreased in starved cells compared to control full-fed cells. The paracellular permeability of urea (molecular radius 2.9 Å), but not mannitol (4.1 Å), inulin (15 Å), and 10-kDa dextran (23 Å) was significantly reduced in starved cells. Treatment of nutrient fed Caco-2 monolayers with pharmacologic autophagy inducers rapamycin and PP242 also caused similar increase in TER comparable to that of control starved Caco-2 manolayers, and reduced paracellular urea flux. On the other hand, autophagy inhibitors bafilomycin A1, chloroquine, and wortmannin attenuated starvation-induced increase in TER. Also, siRNA silencing of autophagy-related protein ATG16L1 and ATG7, but not transfection with non-targeted siRNA, significantly inhibited starvation-induced increase in TER and reduction in paracellular urea flux. Immunoblotting for TJ proteins showed a remarkable decrease in the level of pore forming claudin-2 and an increase in level of occludin after starvation. Starvation did not affect claudin-1,-3,-8, and ZO-1 protein levels. In confocal immunofluorescence, reduced membrane immunostaining and increased cytoplasmic colocalization with lysosome marker LAMP-2 was seen for claudin-2 but not occludin, indicating specific degradation of claudin-2 during induction of autophagy. Conclusion: Overall, our data show that autophagy reduces paracellular TJ permeability of ions and small molecules by down-regulation TJ protein claudin-2.
Su1868 Constant Stimulation of Epithelial Cell Death in the Steady-State Gut Is Controlled via CFLIP Nadine Wittkopf, Claudia Günther, Eva Martini, You-Wen He, Marcus Schuchmann, Markus F. Neurath, Christoph Becker Introduction: The gut is composed of several cell types including intestinal epithelial cells (IECs) and immune cells. The complex interaction between immune cells and intestinal epithelial cells is of utmost importance for gut homeostasis and has been demonstrated to be involved in diseases of the gut such as inflammatory bowel disease, intestinal infections, and colorectal cancer. In the steady-state gut and in the course of gut diseases, intestinal epithelial cells not only signal information about the gut lumen to the subjacent immune cells, most importantly, they respond to mediators produced by immune cells which thereby shape the behavior of IECs in an appropriate way. Methods: Mice with an inducible deletion of cFlip (cellular FLICE-like inhibitory protein) in IECs (cFlipiΔIEC) were generated by crossbreeding cFlipfl mice to Vil-CreERT2 mice. The influence of external cell death triggers in the gut on intestinal epithelial cell death was investigated by organoid culture, endoscopy, immunofluorescence stainings, western blot and qPCR. Results: Deletion of the caspase-8 inhibitor cFlip in intestinal epithelial cells in vivo caused excessive death of IECs in both villi and crypts along with breakdown of intestinal epithelial barrier integrity, inflammation, loss of tissue architecture and ultimately led to death of mice. In striking contrast, deletion of cFlip in crypt epithelial cells in organoid culture in vitro did not result in intestinal epithelial cell death indicating that constant cell death triggers were present in the steadystate gut of cFlipiΔIEC mice but not in organoid culture. Accordingly, death of cFlip deficient organoids was achieved after external addition of cell death receptor ligands such as TNFα (tumor necrosis factor alpha) or CD95L (CD95 ligand). Histological analysis of cFlip deficient organoids demonstrated them to stain highly positive for activated caspase-3 and activated caspase-8 and therefore to die via apoptosis. Interestingly, the cell death mediators present in the steady-state gut and inducing apoptosis in cFlip deficient IECs seemed to be independent from age of mice and probably from the gut flora: cFlip deficient mice died in utero and mice at age 0 weeks, 2 weeks, 5 weeks, and 15 weeks died at similar time points after inducing cflip deficiency and showed comparable IEC death and loss of tissue architecture. Conclusion: Our data suggest the constant presence of cell death triggers in the uninflamed steady-state gut. The activation of intestinal epithelial cell death needs to be tightly regulated to prevent from severe gut dysfunction. cFlip turned out to be a central regulator of IEC behavior responding to the cell death triggers constantly present in the gut.
Su1866 Mast Cell Corticotropin Releasing Factor Receptor 2 Regulates IgE-Mediated Mast Cell Degranulation, Anaphylaxis, and Intestinal Permeability Susan D'Costa, Laura Edwards, Adam J. Moeser The corticotropin releasing factor (CRF) system composed of CRF, related urocortin ligands and receptors CRF1 and CRF2 is the primary regulator of the stress response and a key player in stress-related GI diseases. While the CRF system has been studied extensively in the nervous system, its role in regulating the immune system remains poorly defined. The specific objective of this study was to determine the role of CRF2 in the regulation of immunological responses and intestinal injury induced by IgE-mediated passive systemic anaphylaxis (PSA). To induce PSA, wildtype and CRF2-/- mice were sensitized with antidinitrophenyl (DNP)-IgE overnight followed by administration of DNP-HSA antigen. No significant genotypic differences were found in body temperature and histamine levels prior to antigen injection. Wildtype (WT) and CRF2-/- mice exhibited a similar degree of hypothermia following PSA. However, compared with WT mice, CRF2-/- mice exhibited markedly increased mast cell degranulation indicated by higher serum histamine levels (3.9fold, p<0.05) and histological evaluation. To evaluate the effect of anaphylactic stress on the gut, the jejunum and colon were harvested from mice 120 minutes post-PSA and mounted on the Ussing Chamber for measurements of Transepithelial Electrical Resistance (TER) and 4 kDa-FITC dextran (FD4) flux. Compared to wildtype mice, CRF2-/- mice had increased FD4 permeability across jejunal (41% increase; p<0.05) and colonic mucosa (160%
S-489
AGA Abstracts
AGA Abstracts
investigate the relationship between p53 status of cancer cells and activation of fibroblasts was estimated pathologically using human colorectal cancer tissues. We used HCT116, a cultured cell line derived from human colon cancer tissue: p53-wild type HCT116p53+/+, p53-knockout HCT116p53-/-. Furthermore, we established p53-suppressed cell line using p53 shRNA (HCT116sh p53) compared with HCT116sh control. To elucidate the effect of fibroblast existence on tumor growth, in in vivo study, HCT116 p53+/+ , HCT116 p53-/-, HCT116sh control and HCT116sh p53 were implanted subcutaneously into nude mice, with or without WI-38, a human fibroblast cell line, and the volume of implanted subcutaneous tumors were determined. In in vitro study, the gene expression signature of HCT116 cells co-cultured directly or through transwell filters with or without WI-38 was determined.