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M1824 Inhibitor of Apoptosis Protein 2 (cIAP2) Overexpression in Cytokine Stimulated Colonocytes Modifies Inflammation Driven Propagation of DNA-Damaged Cells
AGA Abstracts
in ileal mucosa was lower in villin-TLR4 than in WT mice. Lysozyme production was also lower in villin-TLR4 Paneth cells than that in WT. In contrast, the percentage of goblet cells per total IECs in each crypt unit was significantly higher in villin-TLR4 mice than in WT mice (13.11±0.36 vs 11.20±0.32%, p<0.01). The number of bacterial colonies growing from stool of WT mice was significantly higher than that in stool from villin-TLR4 in both aerobic and anaerobic cultures. In addition, considerable differences were observed in types of bacteria isolated from stool cultures between villin-TLR4 and WT mice. Conclusions: Epithelial TLR4 signaling positively regulates differentiation of goblet cells but negatively that of Paneth cells. Although Paneth cells are fewer, epithelial expression of TLR4 suppresses fecal bacterial load. These results indicate that TLRs can alter differentiation of cells as they emerge from the stem cell niche.
M1823 Genetic and Functional Analysis of Intestinal Organic Cation/L-Carnitine Transporter (OCTN) in Crohn's Disease Marc Girardin, Philippe Goyette, John D. Rioux, Serge Dionne, Patrick Charlebois, Ernest G. Seidman Background: The IBD5 locus has been repeatedly implicated as a genetic risk factor for Crohn's Disease (CD). This locus codes for the organic cation/carnitine transporters (OCTN1 & 2) that transport cations as well as carnitine, an essential cofactor for long-chain fatty acid oxidation. Two variants in the OCTN cluster have been reported, forming a haplotype associated with susceptibility to CD. They were shown to be associated with modified transporter functions of OCTN1 In Vitro. OCTN1 is a bidirectional transporter, with a low affinity for carnitine compared to OCTN2. The aim of this study is to investigate the functional aspects of intestinal OCTNs in inflammatory bowel disease (IBD) in relation to genetic polymorphisms. Patients & Methods: Intestinal tissue was obtained from endoscopic biopsies and surgical resections in consenting IBD patients (n =33 & 14, resp.) as well from normal controls (n =22 & 14, resp.). Western Blot analysis was used to measure OCTN protein levels in homogenates of intestinal biopsies, using actin as a control. Functional analyses were performed utilizing brush border membrane vesicles isolated from intestinal resections. Carnitine transport was measured across the apical membrane using H3 radiolabeled substrate. Genetic analyses (common OCTN1 & 2 polymorphisms) were carried out using leukocytes. Results: We documented the presence of intestinal OCTN1 & 2 (65 KDa) in both IBD and control patients' tissue by Western blot. OCTN1 protein levels were significantly higher in ileal compared to colonic tissue (2.95% ± 0.4 vs 0.66% ± 0.2, resp.; p<0.0002). OCTN1 expression was found to be higher in CD patients with homo or heterozygous mutations (0.6% ± 0.1 vs 3% ± 0.8, resp., p<0.02). Functional studies showed a very rapid, Na+ dependent transport of carnitine across the apical intestinal border (10 sec). No difference in carnitine transport was found comparing CD and control groups (0.45 ± 0.12 vs 0.51 ± 0.12 nM carnitine/mg prot/min, resp.). Carnitine transport tended to be higher in tissue from patients with homo or heterozygous OCTN1 mutations (0.19 ± 0.03 vs 0.59 ± 0.12, resp.). However, this difference did not reach statistical significance, due to the limited number of cases. Conclusions: The present data reveal the presence of higher OCTN protein levels in intestinal tissue from IBD patients. Our results suggest that ileal carnitine transport is similar in CD and control groups. However, there was a trend towards higher carnitine transport in patients with OCTN1 mutations. Further analyses are underway in a larger cohort to confirm these findings. Supported by a Crohn's and Colitis Foundation of Canada grant.
M1821 Prostaglandin E2 Inhibits Migration of Colonic Lamina Propria Fibroblasts Florian Rieder, Martina Georgieva, Anja Schirbel, Monika Artinger, Anita Zuegner, Martin Blank, Julia Brenmoehl, Jurgen Scholmerich, Gerhard Rogler BACKGROUND: Migration of colonic lamina propria fibroblasts (CLPF) is an important mechanism during wound healing in inflammatory bowel disease (IBD). The concentration of Prostaglandin E2 (PGE2) is increased in the intestinal mucosa of IBD patients. We therefore investigated the role of PGE2 in CLPF migration. METHODS: Primary cultures of CLPF were isolated from healthy controls and Crohn's disease patients. Migration assays were performed in the Boyden chamber and wound scratch assays. Expression of EP-receptors on CLPF, secretion of PGE2, levels of intracellular cAMP, expression and distribution of Factin, α-smooth muscle actin (SMA) and myosin light chain (MLC) were determined by immunoblotting, immunocytochemistry and ELISA, respectively. RESULTS: All four EPreceptor subtypes were present on all tested CLPF. PGE2 and agonists to the EP2- and EP4receptor reduced the migration of CLPF, whereas ligation of the EP1/EP3-receptors did not alter CLPF migration. Blockade of the EP2- and the EP4-receptor inhibited the effect of PGE2 on CLPF migration. Increase in intracellular cAMP by forskolin or phosphodiesterase inhibitors reduced CLPF migration. PGE2 increased the levels of cAMP in CLPF, with abrogation after addition of EP2- and EP4-receptor antagonists. PGE2 and forskolin decreased the expression of α-SMA and F-actin and reduced cell polarization and lamellipodium formation in a wound scratch assay. In addition, forskolin reduced the phoshorylation of MLC (pMLC) and led to lack of accumulation of pMLC in the leading edge of CLPF. Finally CLPF secreted increased amounts of PGE2 after activation with IL-1β. CONCLUSIONS: PGE2 reduced the migration of CLPF via its EP2- and EP4-receptors and elevation of intracellular cAMP. Potential mechanisms are changes in expression of cytoskeletal proteins, failure of CLPF to polarize and a decreased amount of pMLC. CLPF secrete enhanced amounts of PGE2 under inflammatory conditions. This might be one possible reason for the impairment of intestinal wound healing in IBD.
M1824 Inhibitor of Apoptosis Protein 2 (cIAP2) Overexpression in Cytokine Stimulated Colonocytes Modifies Inflammation Driven Propagation of DNADamaged Cells Jakob B. Seidelin, Ole H. Nielsen
M1822
Introduction: Ulcerative colitis (UC) is associated with an increased risk of colorectal cancer, especially in poorly controlled, long-standing, and extensive cases. DNA-instability is an early prerequisite for cancer development. Inhibition of apoptosis pathways lead to survival of DNA-damaged cells and might be of importance for the development of cancer in UC. DNA-strand breaks result in phosphorylation of histone H2A.X which is a part of the cellular DNA-damage surveillance system, and accordingly this phosphorylation can be measured as a surrogate marker of DNA damage. We have previously described that cIAP2 is upregulated in regenerating colonocytes of active UC, but not in quiescent UC [1], and colonocytes have an increased H2A.X phosphorylation in active stages of UC [2]. Aim: The aim was to investigate if cytokines stimulate cIAP2 upregulation in Caco2 cells and lead to increased H2A.X phosphorylation as a sign of DNA-damage. Further, the aim was to investigate if an increased cIAP2 expression prolongs DNA-damage in colonocytes. Methods: Caco2 cells were stimulated with either a combination of cytokines (IL-1β, TNF-α, and INF-γ, all at 1 μM) or doxorubicin (10 μM) for 4 hours. Expression of cIAP2, H2A.X and phospho-H2A.X was determined by immunoblotting at different time points. cIAP2 was inhibited by siRNA interference. Cell death was measured by the LDH-release assay. Results: After 24 hours H2A.X phosphorylation was increased > 2 fold in cells stimulated with cytokines compared to unstimulated controls (p<0.03); the increment was similar to that of doxorubicin treated cells. H2A.X phoshorylation remained high after 72 hours post-stimulation in both cytokine as well as doxorubicin stimulated cells. cIAP2 expression was increased both at mRNA and protein levels in cytokine stimulated cells as compared to controls (p<0.02). Inhibition of cIAP2 expression with siRNA interference caused normalization of H2A.X phosphorylation within 48 hours. cIAP2 inhibition also caused increased cell death in cytokine stimulated colonocytes (p<0.01). Conclusions: Cytokine stimulation of colonocytes in UC promotes DNA-damage at similar levels as doxorubicin, indicating a direct association between proinflammatory mediators and DNA-damage. This DNA-damage is prolonged by a concomitant increased cIAP2 expression, and inhibition of cIAP2 decreases DNA-damage. Thus, cIAP2 might be of importance for DNA-damage propagation and thereby for carcinogenesis observed in UC. References: 1. Seidelin JB et al. Virchows Arch 2007;451:1031 2. Risques RA et al. Gastroenterology 2008;135:410
Lack of Epigenetic Changes Associated With Tolerized Genes During Sustained Inflammatory Gene Expression by Human Intestinal Fibroblasts (HIF) Melania Scarpa, Franco Scaldaferri, Tammy Sadler, Claudio Fiocchi, Eleni Stylianou Background: Tolerance is indispensable to maintenance of intestinal immune homeostasis. Lack of tolerance results in abnormal immune responses eventually leading to prolonged inflammation in the gut, like in IBD. Mucosal tolerance is mediated by immune cells, however, other cells in the mucosa, such as HIF, may not be tolerizable. Our previous studies have shown that HIF fail to become tolerant to bacterial products and pro-inflammatory cytokines. Epigenetic changes, e.g., histone modifications of chromatin have been implicated in regulating tolerance in immune cells by repressing pro-inflammatory gene expression in a cell-, stimulus- and gene-specific manner. Whether the same or other modifications control the expression of genes in HIF is unknown. Aims: To investigate the histone modifications associated with lack of tolerance in HIF. Materials and Methods: THP-1 monocytes and HIF were either exposed to a single dose of the cytokine TNF (responsive cells) or submitted to a classical tolerization protocol in which initial pretreatment of THP-1 or HIF with TNF for 16h was followed by a further exposure for 1 to 6 hours (tolerized cells). IL6 and IL8 mRNA expression levels (qRT-PCR) were assessed. Histone modifications associated with activation (H3S10Ph, H3K4me3, H3Ac) or repression (H3K9me2, H3K27me3) were measured by Chromatin Immunoprecipitation. Results: IL6 and IL8 mRNA levels were upregulated in responsive THP-1 (after a single exposure to TNF), peaking and decreasing rapidly by 2 hours. Repeated exposure to TNF (tolerized THP-1) abrogated induction of the same cytokine mRNA. Conversely, IL6 and IL8 mRNA levels in “tolerized” HIF increased above those of responsive HIF. In THP-1, levels of H3S10Ph, a mark of transcriptionally active genes, paralleled IL6 gene expression, increasing in responsive THP-1 and selectively decreasing in tolerized THP-1. In contrast, H3S10Ph levels were not increased with cytokine gene expression in responsive or “tolerized” HIF. Moreover, decreased H3K9me2 at the IL8 promoter, associated with gene silencing, was detected in responsive but not in tolerized THP-1. Of the other modifications detected in HIF, H3Ac and K3K27me3 were not associated with changes in IL6 gene expression. Conclusions: These preliminary results indicate that whereas decreased H3S10Ph mediates tolerization of inflammatory genes such as IL6 in THP-1 this is not the case in “tolerized” HIF indicating that these cells sustain inflammatory gene expression and potentially gut inflammation in a distinct cell and gene-specific manner. The other types of epigenetic modification that may be involved are currently being investigated.
M1825 Repeated Enemas of Recombinant Human Hepatocyte Growth Factor Ameliorate Rat Experimental Colitis Without Increasing Serum HGF Levels Hitoshi Setoyama, Akio Ido, Toshio Sakiyama, Fumisato Sasaki, Shuji Kanmura, Naohisa Yamaji, Masatsugu Numata, Akihiro Moriuchi, Hirohito Tsubouchi Background and aims: Inflammatory bowel disease (IBD), which includes both ulcerative colitis and Crohn's disease, is an idiopathic and refractory disorder with an unknown etiology. Immune-modulating agents have been used as conventional therapies for IBD. Recently, therapies that regenerate damaged epithelium have been developed as a potential therapeutic modality for IBD. Hepatocyte growth factor (HGF) is a major agent that promotes hepatocyte
AGA Abstracts
S-426
in ileal mucosa was lower in villin-TLR4 than in WT mice. Lysozyme production was also lower in villin-TLR4 Paneth cells than that in WT. In contrast, the percentage of goblet cells per total IECs in each crypt unit was significantly higher in villin-TLR4 mice than in WT mice (13.11±0.36 vs 11.20±0.32%, p<0.01). The number of bacterial colonies growing from stool of WT mice was significantly higher than that in stool from villin-TLR4 in both aerobic and anaerobic cultures. In addition, considerable differences were observed in types of bacteria isolated from stool cultures between villin-TLR4 and WT mice. Conclusions: Epithelial TLR4 signaling positively regulates differentiation of goblet cells but negatively that of Paneth cells. Although Paneth cells are fewer, epithelial expression of TLR4 suppresses fecal bacterial load. These results indicate that TLRs can alter differentiation of cells as they emerge from the stem cell niche.
M1823 Genetic and Functional Analysis of Intestinal Organic Cation/L-Carnitine Transporter (OCTN) in Crohn's Disease Marc Girardin, Philippe Goyette, John D. Rioux, Serge Dionne, Patrick Charlebois, Ernest G. Seidman Background: The IBD5 locus has been repeatedly implicated as a genetic risk factor for Crohn's Disease (CD). This locus codes for the organic cation/carnitine transporters (OCTN1 & 2) that transport cations as well as carnitine, an essential cofactor for long-chain fatty acid oxidation. Two variants in the OCTN cluster have been reported, forming a haplotype associated with susceptibility to CD. They were shown to be associated with modified transporter functions of OCTN1 In Vitro. OCTN1 is a bidirectional transporter, with a low affinity for carnitine compared to OCTN2. The aim of this study is to investigate the functional aspects of intestinal OCTNs in inflammatory bowel disease (IBD) in relation to genetic polymorphisms. Patients & Methods: Intestinal tissue was obtained from endoscopic biopsies and surgical resections in consenting IBD patients (n =33 & 14, resp.) as well from normal controls (n =22 & 14, resp.). Western Blot analysis was used to measure OCTN protein levels in homogenates of intestinal biopsies, using actin as a control. Functional analyses were performed utilizing brush border membrane vesicles isolated from intestinal resections. Carnitine transport was measured across the apical membrane using H3 radiolabeled substrate. Genetic analyses (common OCTN1 & 2 polymorphisms) were carried out using leukocytes. Results: We documented the presence of intestinal OCTN1 & 2 (65 KDa) in both IBD and control patients' tissue by Western blot. OCTN1 protein levels were significantly higher in ileal compared to colonic tissue (2.95% ± 0.4 vs 0.66% ± 0.2, resp.; p<0.0002). OCTN1 expression was found to be higher in CD patients with homo or heterozygous mutations (0.6% ± 0.1 vs 3% ± 0.8, resp., p<0.02). Functional studies showed a very rapid, Na+ dependent transport of carnitine across the apical intestinal border (10 sec). No difference in carnitine transport was found comparing CD and control groups (0.45 ± 0.12 vs 0.51 ± 0.12 nM carnitine/mg prot/min, resp.). Carnitine transport tended to be higher in tissue from patients with homo or heterozygous OCTN1 mutations (0.19 ± 0.03 vs 0.59 ± 0.12, resp.). However, this difference did not reach statistical significance, due to the limited number of cases. Conclusions: The present data reveal the presence of higher OCTN protein levels in intestinal tissue from IBD patients. Our results suggest that ileal carnitine transport is similar in CD and control groups. However, there was a trend towards higher carnitine transport in patients with OCTN1 mutations. Further analyses are underway in a larger cohort to confirm these findings. Supported by a Crohn's and Colitis Foundation of Canada grant.
M1821 Prostaglandin E2 Inhibits Migration of Colonic Lamina Propria Fibroblasts Florian Rieder, Martina Georgieva, Anja Schirbel, Monika Artinger, Anita Zuegner, Martin Blank, Julia Brenmoehl, Jurgen Scholmerich, Gerhard Rogler BACKGROUND: Migration of colonic lamina propria fibroblasts (CLPF) is an important mechanism during wound healing in inflammatory bowel disease (IBD). The concentration of Prostaglandin E2 (PGE2) is increased in the intestinal mucosa of IBD patients. We therefore investigated the role of PGE2 in CLPF migration. METHODS: Primary cultures of CLPF were isolated from healthy controls and Crohn's disease patients. Migration assays were performed in the Boyden chamber and wound scratch assays. Expression of EP-receptors on CLPF, secretion of PGE2, levels of intracellular cAMP, expression and distribution of Factin, α-smooth muscle actin (SMA) and myosin light chain (MLC) were determined by immunoblotting, immunocytochemistry and ELISA, respectively. RESULTS: All four EPreceptor subtypes were present on all tested CLPF. PGE2 and agonists to the EP2- and EP4receptor reduced the migration of CLPF, whereas ligation of the EP1/EP3-receptors did not alter CLPF migration. Blockade of the EP2- and the EP4-receptor inhibited the effect of PGE2 on CLPF migration. Increase in intracellular cAMP by forskolin or phosphodiesterase inhibitors reduced CLPF migration. PGE2 increased the levels of cAMP in CLPF, with abrogation after addition of EP2- and EP4-receptor antagonists. PGE2 and forskolin decreased the expression of α-SMA and F-actin and reduced cell polarization and lamellipodium formation in a wound scratch assay. In addition, forskolin reduced the phoshorylation of MLC (pMLC) and led to lack of accumulation of pMLC in the leading edge of CLPF. Finally CLPF secreted increased amounts of PGE2 after activation with IL-1β. CONCLUSIONS: PGE2 reduced the migration of CLPF via its EP2- and EP4-receptors and elevation of intracellular cAMP. Potential mechanisms are changes in expression of cytoskeletal proteins, failure of CLPF to polarize and a decreased amount of pMLC. CLPF secrete enhanced amounts of PGE2 under inflammatory conditions. This might be one possible reason for the impairment of intestinal wound healing in IBD.
M1824 Inhibitor of Apoptosis Protein 2 (cIAP2) Overexpression in Cytokine Stimulated Colonocytes Modifies Inflammation Driven Propagation of DNADamaged Cells Jakob B. Seidelin, Ole H. Nielsen
M1822
Introduction: Ulcerative colitis (UC) is associated with an increased risk of colorectal cancer, especially in poorly controlled, long-standing, and extensive cases. DNA-instability is an early prerequisite for cancer development. Inhibition of apoptosis pathways lead to survival of DNA-damaged cells and might be of importance for the development of cancer in UC. DNA-strand breaks result in phosphorylation of histone H2A.X which is a part of the cellular DNA-damage surveillance system, and accordingly this phosphorylation can be measured as a surrogate marker of DNA damage. We have previously described that cIAP2 is upregulated in regenerating colonocytes of active UC, but not in quiescent UC [1], and colonocytes have an increased H2A.X phosphorylation in active stages of UC [2]. Aim: The aim was to investigate if cytokines stimulate cIAP2 upregulation in Caco2 cells and lead to increased H2A.X phosphorylation as a sign of DNA-damage. Further, the aim was to investigate if an increased cIAP2 expression prolongs DNA-damage in colonocytes. Methods: Caco2 cells were stimulated with either a combination of cytokines (IL-1β, TNF-α, and INF-γ, all at 1 μM) or doxorubicin (10 μM) for 4 hours. Expression of cIAP2, H2A.X and phospho-H2A.X was determined by immunoblotting at different time points. cIAP2 was inhibited by siRNA interference. Cell death was measured by the LDH-release assay. Results: After 24 hours H2A.X phosphorylation was increased > 2 fold in cells stimulated with cytokines compared to unstimulated controls (p<0.03); the increment was similar to that of doxorubicin treated cells. H2A.X phoshorylation remained high after 72 hours post-stimulation in both cytokine as well as doxorubicin stimulated cells. cIAP2 expression was increased both at mRNA and protein levels in cytokine stimulated cells as compared to controls (p<0.02). Inhibition of cIAP2 expression with siRNA interference caused normalization of H2A.X phosphorylation within 48 hours. cIAP2 inhibition also caused increased cell death in cytokine stimulated colonocytes (p<0.01). Conclusions: Cytokine stimulation of colonocytes in UC promotes DNA-damage at similar levels as doxorubicin, indicating a direct association between proinflammatory mediators and DNA-damage. This DNA-damage is prolonged by a concomitant increased cIAP2 expression, and inhibition of cIAP2 decreases DNA-damage. Thus, cIAP2 might be of importance for DNA-damage propagation and thereby for carcinogenesis observed in UC. References: 1. Seidelin JB et al. Virchows Arch 2007;451:1031 2. Risques RA et al. Gastroenterology 2008;135:410
Lack of Epigenetic Changes Associated With Tolerized Genes During Sustained Inflammatory Gene Expression by Human Intestinal Fibroblasts (HIF) Melania Scarpa, Franco Scaldaferri, Tammy Sadler, Claudio Fiocchi, Eleni Stylianou Background: Tolerance is indispensable to maintenance of intestinal immune homeostasis. Lack of tolerance results in abnormal immune responses eventually leading to prolonged inflammation in the gut, like in IBD. Mucosal tolerance is mediated by immune cells, however, other cells in the mucosa, such as HIF, may not be tolerizable. Our previous studies have shown that HIF fail to become tolerant to bacterial products and pro-inflammatory cytokines. Epigenetic changes, e.g., histone modifications of chromatin have been implicated in regulating tolerance in immune cells by repressing pro-inflammatory gene expression in a cell-, stimulus- and gene-specific manner. Whether the same or other modifications control the expression of genes in HIF is unknown. Aims: To investigate the histone modifications associated with lack of tolerance in HIF. Materials and Methods: THP-1 monocytes and HIF were either exposed to a single dose of the cytokine TNF (responsive cells) or submitted to a classical tolerization protocol in which initial pretreatment of THP-1 or HIF with TNF for 16h was followed by a further exposure for 1 to 6 hours (tolerized cells). IL6 and IL8 mRNA expression levels (qRT-PCR) were assessed. Histone modifications associated with activation (H3S10Ph, H3K4me3, H3Ac) or repression (H3K9me2, H3K27me3) were measured by Chromatin Immunoprecipitation. Results: IL6 and IL8 mRNA levels were upregulated in responsive THP-1 (after a single exposure to TNF), peaking and decreasing rapidly by 2 hours. Repeated exposure to TNF (tolerized THP-1) abrogated induction of the same cytokine mRNA. Conversely, IL6 and IL8 mRNA levels in “tolerized” HIF increased above those of responsive HIF. In THP-1, levels of H3S10Ph, a mark of transcriptionally active genes, paralleled IL6 gene expression, increasing in responsive THP-1 and selectively decreasing in tolerized THP-1. In contrast, H3S10Ph levels were not increased with cytokine gene expression in responsive or “tolerized” HIF. Moreover, decreased H3K9me2 at the IL8 promoter, associated with gene silencing, was detected in responsive but not in tolerized THP-1. Of the other modifications detected in HIF, H3Ac and K3K27me3 were not associated with changes in IL6 gene expression. Conclusions: These preliminary results indicate that whereas decreased H3S10Ph mediates tolerization of inflammatory genes such as IL6 in THP-1 this is not the case in “tolerized” HIF indicating that these cells sustain inflammatory gene expression and potentially gut inflammation in a distinct cell and gene-specific manner. The other types of epigenetic modification that may be involved are currently being investigated.
M1825 Repeated Enemas of Recombinant Human Hepatocyte Growth Factor Ameliorate Rat Experimental Colitis Without Increasing Serum HGF Levels Hitoshi Setoyama, Akio Ido, Toshio Sakiyama, Fumisato Sasaki, Shuji Kanmura, Naohisa Yamaji, Masatsugu Numata, Akihiro Moriuchi, Hirohito Tsubouchi Background and aims: Inflammatory bowel disease (IBD), which includes both ulcerative colitis and Crohn's disease, is an idiopathic and refractory disorder with an unknown etiology. Immune-modulating agents have been used as conventional therapies for IBD. Recently, therapies that regenerate damaged epithelium have been developed as a potential therapeutic modality for IBD. Hepatocyte growth factor (HGF) is a major agent that promotes hepatocyte
AGA Abstracts
S-426