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Mo1835 Investigation of Slow Cycling Stem Cells and Paneth Cells in T. Spiralis-Induced Small Intestinal Inflammation
AGA Abstracts
greatly diminished growth of the pathogen was shown in these organs of CAC-mice treated with CCL1 antisense ODN. Conclusion: M1Mϕ were induced in CAC-mice by CCL1 antisense ODN and these mice were survived after E.faecalis infection. These results indicate the sepsis stemming from E.faecalis translocation in CAC-mice is controllable by CCL1 antisense ODN gene therapy.
Atoh1 was disappeared in the nuclei of HD6 negative crypts. Conclusion:Both TCF4/βcatenin protein and Atoh1 are essential to express HD6 in different from HD5. The decrease of HD6 by the suppression ofβ-catenin in small intestine might cause mucosal barrier dysfunction, suggesting that HD6 might be one of the pathogenesis of CD. Mo1834
Mo1832 NOD2 Downregulates Colonic Inflammation Through IRF-4 Mediated Inhibition of K63 Directed Polyubiquitination of RICK and TRAF6 Molecules Tomohiro Watanabe, Naoki Asano, atsushi kitani, Ivan J. Fuss, Warren Strober
In Vivo Animal Model to Study Antimicrobial Therapy of C. difficile Infection Zhi-Dong Jiang, Shi Ke Recommended therapy of bacterial infection is generally based on in vitro susceptibility of an organism to an infecting organism. Yet drug bioavailability based on solubility, protein binding and other factors along with host resistance and inflammatory-immune response importantly alter response to treatment. The objective of the study was to establish in vivo animal model to study antimicrobial therapy of C. difficile infection. C. difficile isolate with hyper-virulent factors (toxin A/B plus binary toxin and 18 base pair deletion in tcdC gene) was used in the study. The cultured C. difficile was stained with near infrared (NIR) dye. Stained C. difficile were determined by using fluorescent microscope and confocal microscope. To monitor stained bacterial trafficking in the gut animals were imaged in real-time. The intestines from treated animals were dissected at the end of imaging study. The bacterial signal intensity and migration pattern were recorded and compared. C. difficile toxin secretion in the different segment of GI system was analyzed. Results showed the C. difficile can be stained by NIR dye. Stained bacteria can be imaged by NIR camera system to track the C. difficile traffic in the gut in real-time. Untreated control animal has different bacteria migration pattern than the animal with C. difficile infection treated with ciprofloxacin. The NIR florescent probe can be used to visualize C. difficile trafficking in the gastrointentinal tract through culturing, staining and imaging. Fig. 1. Confocal images of C. difficile. Viable bacterial stained with NIR dye (A. Red) and dead bacterial determined with Sytox green (B. Green). Stain efficiency was determined by co-localized all cells (C. Green) and NIR (C. Red). Confocal microscope analyzed NIR dye binding at subcellular level (D). Red and green represent NIR and cell nuclei, respectively. Results demonstrate C. difficile can be stained by NIR dye. Most bacteria remain viable after the staining procedure.
Background: It has been established that polymorphisms of the caspase activation and recruitment domain 15 (CARD15), a major risk factor in Crohn's disease (CD), lead to loss of nucleotide-binding oligomerization domain 2 (NOD2) function. However, a molecular explanation of how such loss of function leads to increased susceptibility to CD has remained unclear. Aim: In a previous study exploring this question, we reported that activation of NOD2 in human dendritic cells by its ligand, muramyl dipeptide (MDP), negatively regulates Toll-like receptor (TLR)-mediated inflammatory responses to intestinal bacterial components (ie MDP, LPS) through the generation of Interferon Regulating Factor 4 (IRF4). However, the precise mode of regulation remained unclear. Results: In the present studies, we show that NOD2 activation results in increased interferon regulatory factor 4 (IRF4) expression and subsequent binding to tumor necrosis factor receptor associated factor 6 (TRAF6) and RICK (receptor interacting serine-threonine kinase). We then found that such binding leads to IRF4-mediated inhibition of Lys63-linked polyubiquitination of TRAF6 and RICK and to subsequent downregulation of nuclear factor (NF)-κB activation. Finally, we demonstrate that protection of mice from the development of experimental colitis by MDP or IRF4 administration is accompanied by similar IRF4-mediated effects on polyubiquitination of TRAF6 and RICK in colonic lamina propria mononuclear cells. Conclusion: These findings thus define a mechanism of NOD2-mediated regulation of innate immune responses to intestinal microflora components that could explain the relation of CARD15 polymorphisms and resultant NOD2 dysfunction to CD. Mo1835 Investigation of Slow Cycling Stem Cells and Paneth Cells in T. SpiralisInduced Small Intestinal Inflammation Tanvi Javkar, Fred Sablitzky, Yashwant R. Mahida Introduction: In the small intestine (SI), two populations of stem cells have been characterized, those intercalated between Paneth cells and slow cycling (quiescent) stem cells above the Paneth cell zone (around cell position 4). Our previous studies have shown that the latter cells proliferate during epithelial regeneration post-irradiation. The aim of this study was to investigate changes in slow cycling stem cells and Paneth cells in the T. spiralisinfection model of T cell-mediated inflammation in SI. Methods: Transgenic mice, in which doxycycline induces expression of histone 2B (H2B)-green fluorescent protein (GFP), were used. Following discontinuation of doxycycline ("chase" period), retention of H2B-GFP enables the identification of slow cycling stem cells and long-lived Paneth cells. Inflammation in SI was induced by oral administration of T. spiralis larvae and experiments were designed such that mucosal samples were obtained on day 13 of infection, which coincided with day 14 of post-doxycycline chase period. Epithelial retention of H2B-GFP per crypt cell position (cp) was studied following immunohistochemistry and using Score and Wincrypts program. For cells of interest, percentage at a particular crypt cp is expressed as the labelling index (LI). Whole crypt LI was also determined. Data are presented as median (IQR). Results: H2B-GFP-retaining stem cells: pooled data for all regions of the SI showed significant reduction in the number of H2B-GFP-retaining stem cells in T. spiralis-infected sections compared to non-infected controls [0.2 (0 - 0.7)] vs 0.9 (0.3 - 1.4); p<0.0001]. Data for different regions of the SI are given in Table. H2B-GFP-retaining stem cells peaked at around cp 4 in controls sections, but smaller peaks at higher cell positions (>10) were seen in sections of inflamed SI. Thus, in all regions of SI, LI for cp 2-6 was significantly (p<0.01) higher in controls compared to infected samples. Paneth cells: in the inflamed SI, there was significant increase in the total number of Paneth cells [whole SI: 26.6 (24.3 - 30.0) vs 16.3 (14.0 - 19.1); p<0.0001; also see Table), but significant reduction in H2B-GFP-retaining Paneth cells (whole SI: 3.0 (1.2 - 4.8) vs 6.5 (4.6 - 7.4); p<0.0001; also see Table). Conclusions: In T. spiralis-induced inflammation in SI, the increase in the total number of Paneth cells but reduction in H2B-GFP-retaining Paneth cells suggests not only increased turnover of these cells, but also stem cell differentiation along the secretory pathway. Reduction in the number of H2B-GFP-retaining stem cells in the inflamed SI implies that they divide and preferentially differentiate into Paneth cells. The increase in the total number of Paneth cells at the crypt base leads to a change in the position of the remaining slow cycling stem cells in the T. spiralis-infected SI. Mean (IQR) values for H2B-GFP-retaining stem cells, H2B-GFP-retaining Paneth cells and total Paneth cells in different regions of control and T. spiralis-infected small intestine.
Mo1833 Human Alpha-Defensin 6 Regulated by the Cooperation of Beta-Catenin and ATOH1, Might Be the Pathogenesis of Crohn's Disease Ryohei Hayashi, Kiichiro Tsuchiya, Shuji Hibiya, Keita Fukushima, Nobukatsu Horita, Eriko Okada, Akihiro Araki, Kazuo Ohtsuka, Mamoru Watanabe Background and Aims:Antimicrobial mucosal barrier dysfunction, including the reduction of Human alpha-Defensin (HD) 5, is one of the most crucial pathogenesis of Crohn's disease (CD). Human Paneth cells produce two α-defensin peptides, which called HD5 and HD6. Recently, it has been reported that HD6 promotes mucosal innate immunity through selfassembled peptide "nanonets" whereas HD5 has the antimicrobial activity. The transcriptional regulation of HD6 has not been elucidated. Moreover the association of HD6 expression with CD also remains unknown. We therefore aimed to elucidate the transcriptional regulation of HD6 and the pathogenesis of CD by HD6 expression. Methods:To investigate HD6 expression, we transgened Atoh1 into colon cancer cell line; SW480 by lentivirus infection. The expression of HD6 was assessed by quantitative RT-PCR. The transcriptional activity of HD6 promoter was assessed by the luciferase reporter assay. For the analysis of the HD6 expression in CD, non-inflamed jejunum biopsy specimens of 15 CD patients and 9 healthy controls using double balloon endoscopy (DBE) were assessed. Results:HD6 was markedly increased by Atoh1 expression in SW480 whereas other antimicrobial peptides were not changed. Atoh1 also enhanced the transcriptional activity of HD6 promoter. We found that HD6 promoter within 250-bp from ATG contains a transcription factor (TCF) binding site and four E-box binding site. The deletion of each binding sites revealed that not only TCF4/ β-catenin protein complex but also Atoh1 is indispensable for HD6 expression. ChIP assay showed that Atoh1 directly binds to the promoter region of HD6. Moreover, immunostaining showed thatβ-catenin and Atoh1 are co-expressed in the nuclei of HD6 positive cells. Finally, we assessed the HD6 expression in non-inflamed mucosa CD. The microarray using mapping biopsy of entire small intestine in 4 CD patients and 4 non-IBD healthy controls showed that a number of inflammatory related genes were increased in not only terminal ileum but also proximal ileum of CD patients even if any intestinal erosions and ulcers has not been shown in proximal ileum by DBE. On the other hand, almost inflammatory related genes were not shown in jejunum. These results suggested that analysis using jejunum biopsy specimen could rule out the effect of inflammation. Immunostaining showed that HD6 positive Paneth cells in jejunum of CD patients were significantly lower than that of healthy controls. Moreover, HD6 negative crypts were found in only CD patients.β-catenin but not
AGA Abstracts
S-722
greatly diminished growth of the pathogen was shown in these organs of CAC-mice treated with CCL1 antisense ODN. Conclusion: M1Mϕ were induced in CAC-mice by CCL1 antisense ODN and these mice were survived after E.faecalis infection. These results indicate the sepsis stemming from E.faecalis translocation in CAC-mice is controllable by CCL1 antisense ODN gene therapy.
Atoh1 was disappeared in the nuclei of HD6 negative crypts. Conclusion:Both TCF4/βcatenin protein and Atoh1 are essential to express HD6 in different from HD5. The decrease of HD6 by the suppression ofβ-catenin in small intestine might cause mucosal barrier dysfunction, suggesting that HD6 might be one of the pathogenesis of CD. Mo1834
Mo1832 NOD2 Downregulates Colonic Inflammation Through IRF-4 Mediated Inhibition of K63 Directed Polyubiquitination of RICK and TRAF6 Molecules Tomohiro Watanabe, Naoki Asano, atsushi kitani, Ivan J. Fuss, Warren Strober
In Vivo Animal Model to Study Antimicrobial Therapy of C. difficile Infection Zhi-Dong Jiang, Shi Ke Recommended therapy of bacterial infection is generally based on in vitro susceptibility of an organism to an infecting organism. Yet drug bioavailability based on solubility, protein binding and other factors along with host resistance and inflammatory-immune response importantly alter response to treatment. The objective of the study was to establish in vivo animal model to study antimicrobial therapy of C. difficile infection. C. difficile isolate with hyper-virulent factors (toxin A/B plus binary toxin and 18 base pair deletion in tcdC gene) was used in the study. The cultured C. difficile was stained with near infrared (NIR) dye. Stained C. difficile were determined by using fluorescent microscope and confocal microscope. To monitor stained bacterial trafficking in the gut animals were imaged in real-time. The intestines from treated animals were dissected at the end of imaging study. The bacterial signal intensity and migration pattern were recorded and compared. C. difficile toxin secretion in the different segment of GI system was analyzed. Results showed the C. difficile can be stained by NIR dye. Stained bacteria can be imaged by NIR camera system to track the C. difficile traffic in the gut in real-time. Untreated control animal has different bacteria migration pattern than the animal with C. difficile infection treated with ciprofloxacin. The NIR florescent probe can be used to visualize C. difficile trafficking in the gastrointentinal tract through culturing, staining and imaging. Fig. 1. Confocal images of C. difficile. Viable bacterial stained with NIR dye (A. Red) and dead bacterial determined with Sytox green (B. Green). Stain efficiency was determined by co-localized all cells (C. Green) and NIR (C. Red). Confocal microscope analyzed NIR dye binding at subcellular level (D). Red and green represent NIR and cell nuclei, respectively. Results demonstrate C. difficile can be stained by NIR dye. Most bacteria remain viable after the staining procedure.
Background: It has been established that polymorphisms of the caspase activation and recruitment domain 15 (CARD15), a major risk factor in Crohn's disease (CD), lead to loss of nucleotide-binding oligomerization domain 2 (NOD2) function. However, a molecular explanation of how such loss of function leads to increased susceptibility to CD has remained unclear. Aim: In a previous study exploring this question, we reported that activation of NOD2 in human dendritic cells by its ligand, muramyl dipeptide (MDP), negatively regulates Toll-like receptor (TLR)-mediated inflammatory responses to intestinal bacterial components (ie MDP, LPS) through the generation of Interferon Regulating Factor 4 (IRF4). However, the precise mode of regulation remained unclear. Results: In the present studies, we show that NOD2 activation results in increased interferon regulatory factor 4 (IRF4) expression and subsequent binding to tumor necrosis factor receptor associated factor 6 (TRAF6) and RICK (receptor interacting serine-threonine kinase). We then found that such binding leads to IRF4-mediated inhibition of Lys63-linked polyubiquitination of TRAF6 and RICK and to subsequent downregulation of nuclear factor (NF)-κB activation. Finally, we demonstrate that protection of mice from the development of experimental colitis by MDP or IRF4 administration is accompanied by similar IRF4-mediated effects on polyubiquitination of TRAF6 and RICK in colonic lamina propria mononuclear cells. Conclusion: These findings thus define a mechanism of NOD2-mediated regulation of innate immune responses to intestinal microflora components that could explain the relation of CARD15 polymorphisms and resultant NOD2 dysfunction to CD. Mo1835 Investigation of Slow Cycling Stem Cells and Paneth Cells in T. SpiralisInduced Small Intestinal Inflammation Tanvi Javkar, Fred Sablitzky, Yashwant R. Mahida Introduction: In the small intestine (SI), two populations of stem cells have been characterized, those intercalated between Paneth cells and slow cycling (quiescent) stem cells above the Paneth cell zone (around cell position 4). Our previous studies have shown that the latter cells proliferate during epithelial regeneration post-irradiation. The aim of this study was to investigate changes in slow cycling stem cells and Paneth cells in the T. spiralisinfection model of T cell-mediated inflammation in SI. Methods: Transgenic mice, in which doxycycline induces expression of histone 2B (H2B)-green fluorescent protein (GFP), were used. Following discontinuation of doxycycline ("chase" period), retention of H2B-GFP enables the identification of slow cycling stem cells and long-lived Paneth cells. Inflammation in SI was induced by oral administration of T. spiralis larvae and experiments were designed such that mucosal samples were obtained on day 13 of infection, which coincided with day 14 of post-doxycycline chase period. Epithelial retention of H2B-GFP per crypt cell position (cp) was studied following immunohistochemistry and using Score and Wincrypts program. For cells of interest, percentage at a particular crypt cp is expressed as the labelling index (LI). Whole crypt LI was also determined. Data are presented as median (IQR). Results: H2B-GFP-retaining stem cells: pooled data for all regions of the SI showed significant reduction in the number of H2B-GFP-retaining stem cells in T. spiralis-infected sections compared to non-infected controls [0.2 (0 - 0.7)] vs 0.9 (0.3 - 1.4); p<0.0001]. Data for different regions of the SI are given in Table. H2B-GFP-retaining stem cells peaked at around cp 4 in controls sections, but smaller peaks at higher cell positions (>10) were seen in sections of inflamed SI. Thus, in all regions of SI, LI for cp 2-6 was significantly (p<0.01) higher in controls compared to infected samples. Paneth cells: in the inflamed SI, there was significant increase in the total number of Paneth cells [whole SI: 26.6 (24.3 - 30.0) vs 16.3 (14.0 - 19.1); p<0.0001; also see Table), but significant reduction in H2B-GFP-retaining Paneth cells (whole SI: 3.0 (1.2 - 4.8) vs 6.5 (4.6 - 7.4); p<0.0001; also see Table). Conclusions: In T. spiralis-induced inflammation in SI, the increase in the total number of Paneth cells but reduction in H2B-GFP-retaining Paneth cells suggests not only increased turnover of these cells, but also stem cell differentiation along the secretory pathway. Reduction in the number of H2B-GFP-retaining stem cells in the inflamed SI implies that they divide and preferentially differentiate into Paneth cells. The increase in the total number of Paneth cells at the crypt base leads to a change in the position of the remaining slow cycling stem cells in the T. spiralis-infected SI. Mean (IQR) values for H2B-GFP-retaining stem cells, H2B-GFP-retaining Paneth cells and total Paneth cells in different regions of control and T. spiralis-infected small intestine.
Mo1833 Human Alpha-Defensin 6 Regulated by the Cooperation of Beta-Catenin and ATOH1, Might Be the Pathogenesis of Crohn's Disease Ryohei Hayashi, Kiichiro Tsuchiya, Shuji Hibiya, Keita Fukushima, Nobukatsu Horita, Eriko Okada, Akihiro Araki, Kazuo Ohtsuka, Mamoru Watanabe Background and Aims:Antimicrobial mucosal barrier dysfunction, including the reduction of Human alpha-Defensin (HD) 5, is one of the most crucial pathogenesis of Crohn's disease (CD). Human Paneth cells produce two α-defensin peptides, which called HD5 and HD6. Recently, it has been reported that HD6 promotes mucosal innate immunity through selfassembled peptide "nanonets" whereas HD5 has the antimicrobial activity. The transcriptional regulation of HD6 has not been elucidated. Moreover the association of HD6 expression with CD also remains unknown. We therefore aimed to elucidate the transcriptional regulation of HD6 and the pathogenesis of CD by HD6 expression. Methods:To investigate HD6 expression, we transgened Atoh1 into colon cancer cell line; SW480 by lentivirus infection. The expression of HD6 was assessed by quantitative RT-PCR. The transcriptional activity of HD6 promoter was assessed by the luciferase reporter assay. For the analysis of the HD6 expression in CD, non-inflamed jejunum biopsy specimens of 15 CD patients and 9 healthy controls using double balloon endoscopy (DBE) were assessed. Results:HD6 was markedly increased by Atoh1 expression in SW480 whereas other antimicrobial peptides were not changed. Atoh1 also enhanced the transcriptional activity of HD6 promoter. We found that HD6 promoter within 250-bp from ATG contains a transcription factor (TCF) binding site and four E-box binding site. The deletion of each binding sites revealed that not only TCF4/ β-catenin protein complex but also Atoh1 is indispensable for HD6 expression. ChIP assay showed that Atoh1 directly binds to the promoter region of HD6. Moreover, immunostaining showed thatβ-catenin and Atoh1 are co-expressed in the nuclei of HD6 positive cells. Finally, we assessed the HD6 expression in non-inflamed mucosa CD. The microarray using mapping biopsy of entire small intestine in 4 CD patients and 4 non-IBD healthy controls showed that a number of inflammatory related genes were increased in not only terminal ileum but also proximal ileum of CD patients even if any intestinal erosions and ulcers has not been shown in proximal ileum by DBE. On the other hand, almost inflammatory related genes were not shown in jejunum. These results suggested that analysis using jejunum biopsy specimen could rule out the effect of inflammation. Immunostaining showed that HD6 positive Paneth cells in jejunum of CD patients were significantly lower than that of healthy controls. Moreover, HD6 negative crypts were found in only CD patients.β-catenin but not
AGA Abstracts
S-722