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Mo2049 Repeated Cycles of DSS Inducing a Chronically Relapsing Inflammation: A Novel Model to Study Fibrosis Using In Vivo MRI T2 Relaxometry
AGA Abstracts
which is a glucosinolate precursor of SFN, was orally administered to the mice, at dose of 8.5 mg/kg, before and after the injection of IND. SGS is converted to biologically active SFN in the intestinal lumen by myrosinase activity in the intestinal microflora. Expression of hemoxygenase-1 (HO-1) was evaluated by real time RT-PCR. Vascular permeability was assessed by Evans Blue exudation into the mucosa after i.v. injection. Neutrophil activation was evaluated by myeloperoxidase (MPO) activity. Results: 1. IND treatment caused mucosal injury in small intestine, accompanied by the increases in the vascular permeability, the mucosal MPO activity, and the mucosal invasion of anaerobic enterobacteria. 2.SGS attenuated the IND-induced small intestinal injury, and prevented the increases in the vascular permeability and the MPO activity induced by IND. 3. SGS enhanced HO-1 expression in the small intestine, and prevented the increase in mucosal invasion of the anaerobic enterobacteria caused by IND. Conclusions: These results suggest that (1) orally administered SGS protects small intestine from IND-induced injury in mice In Vivo, and that (2) SGS affords protection of the small intestinal mucosa against IND-induced injury, partly by inducing nrf2-dependent antioxidant enzymes in the mucosa, and partly by inhibiting invasion of the anaerobic enterobacteria into the mucosa.
resistant to inflammation, but at later stage exhibited increased dysplasia and proliferation, suggesting its dual role in the course of the disease. Conclusion: The data suggest that the MMP-9 initially mediates inflammation but later exerts a protective role, implying that function of MMP-9 changes as inflammation advances in CR-mediated colitis. Mo2049 Repeated Cycles of DSS Inducing a Chronically Relapsing Inflammation: A Novel Model to Study Fibrosis Using In Vivo MRI T2 Relaxometry Christine M. Breynaert, Tom Dresselaers, Jonathan Cremer, Kristel Van Steen, Clémentine Perrier, Severine Vermeire, Paul J. Rutgeerts, Jan L. Ceuppens, Uwe Himmelreich, Gert Van Assche BACKGROUND Most experimental animal models of IBD fail to accurately reflect the chronically relapsing inflammation underlying the complications of human Crohn's disease. This study investigated whether repeated cycles of DSS adequately reflect the effects of chronic transmural healing. Transmural μMR imaging with In Vivo T2 relaxometry was used to differentiate transmural changes. METHODS DSS colitis was induced in 6 week-old C57BL6/J mice: acute colitis mice (n=10) received 7 days of DSS prior to sacrifice. One cycle mice (n=10) received 1 cycle of 7 days of DSS followed by 2 weeks of normal drinking water prior to sacrifice, 2-cycle mice (n=10) 2 cycles and 3-cycle mice (n=9) 3 cycles prior to sacrifice. Control mice (n=7) received normal drinking water only. Six mice per group were scanned In Vivo on a 9.4T MRI Bruker system. T2 weighted images and T2 maps of the distal colon were recorded. Histograms of the colon wall were created from T2 maps using a plugin for ImageJ. After scanning and euthanasia, the distal colon of all mice was harvested for histology and FACS analysis. Collagen deposition was quantified with MartiusScarlett-Blue staining. RESULTS Colon weight, colon weight/length ratio and macroscopic score were significantly higher in the 1-, 2- and 3-cycle group compared to the control group (p<0.001), however, no significant difference was observed within the cycling groups. Although all mice in the 2-, and 3-cycle group had a normal DAI score, the macroscopic score was significantly higher compared to the acute group (p<0.001). CD4+Foxp3+ cells in blood increased with more cycles of DSS (p<0.001). The number of CD4+Foxp3+ cells in MLN was higher in the 2- and 3-cycle model compared to the acute model and controls (p<0.001). A higher number of IFNg+ cells in MLN was observed in the 1-, 2- and 3-cycle model compared to the acute model p<0.001). The 3-cycle model showed a higher number of IL17+ cells in MLN compared to the acute model and controls (p=0.0012). No significant difference was observed within the different models in IL13. Collagen deposition was significantly higher in the 2- and 3-cycle model compared to the 1-cycle model (p<0.001). T2 mapping of the colon was able to discern between all groups: the increasing number of cycles correlated with a gradual regression of T2 values to those of normal colon. CONCLUSION The immune profile of a cycling DSS model with induction of relapse and remission is clearly different from the acute DSS model inducing an adaptive immune response. The chronic repeated cycles of DSS model opens perspectives to study the effects of healing and fibrosis in a murine model of IBD. In Vivo T2 relaxometry is a promising non-invasive assessment of inflammation and fibrosis and should be explored in CD patients.
Mo2047 Hydrogen Sulfide Synthesis During Colonic Inflammation Occurs Primarily via a Pyridoxal-5'-Phosphate (P5p)-Independent Pathway Kyle L. Flannigan, John L. Wallace Hydrogen sulfide (H2S) is an important endogenous anti-inflammatory mediator, which has been shown to promote resolution of colitis and healing of ulcers. H2S synthesis was believed to occur primarily via two P5P-dependent enzymes (cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE)). In the present study, we examined the possible contribution of a third pathway for H2S synthesis, in the healthy and inflamed colon, which involves the enzymes cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3MST). Methods: The capacity of tissue to produce H2S was measured from homogenized tissue in the presence of exogenous substrate and/or inhibitors using a zinc-trapping assay. Production of H2S via the CAT-3MST pathway was explored using the substrate α-ketoglutarate and the CAT inhibitors L-aspartate and O-carboxymethyl-hydroxylamine hemihydrochloride (CHH). Colitis was induced in rats by intracolonic administration of dinitrobenzene sulfonic acid (DNBS). Rats were euthanized 3 to 28 days post-DNBS for measurement of H2S synthesis, and for immunofluorescent staining to localize CAT and 3MST expression. Results: Colonic H2S synthesis via the CAT-3MST pathway was 5-fold greater than that via the P5P-dependent (CBS and CSE) pathways (225 ± 59 vs. 42 ± 21 nmol/g/h respectively; p<0.05). In the inflamed colon, H2S synthesis via CAT-3MST was markedly increased (>4-fold) compared to controls (964 ± 115 vs. 225 ± 59 nmol/g/h, respectively; p<0.05). Colonic H2S synthesis was greatest when inflammation was most robust (3 days post-DNBS; 1132 ± 88 nmol/g/ h) and was suppressed by L-aspartate and CHH (424 ± 153 and 69 ± 34 nmol/g/h, respectively; both p<0.005). Immunofluorescence staining revealed that CAT and 3MST were colocalized in the colonic epithelium. Moreover, CAT expression was greatest in epithelial cells adjacent to ulcers (MFI: 1202 ± 63 U vs. 873 ± 105 U in healthy epithelium; p<0.05). By day 28, when colitis had largely resolved, H2S synthesis had decreased to control levels. Conclusions: These data suggest that in both the healthy and inflamed colon CAT-3MST represents the primary pathway for H2S synthesis. Increased CAT expression may account for the increased H2S synthesis during inflammation. Given the ability of H2S to promote resolution of inflammation and healing of mucosal injury, understanding the regulation of intestinal H2S synthesis may have important implications for the development of novel therapies for inflammatory bowel disease. Mo2048 Dual Roles Played by Matrix Metalloproteinase (MMP9) in Citrobacter rodentium-induced Hemorrhagic Colitis Sabrina Jeppsson, Maiko Sasaki, Jan-Michael A. Klapproth, Christopher W. Harper, Didier Merlin, Shanthi V. Sitaraman, Pallavi Garg Background and Aims: Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are well known causative agents of diarrheal disease in humans. EPEC is responsible for infantile diarrhea whereas EHEC causes hemorrhagic colitis. The molecular pathogenesis of EPEC and EHEC related infections have been well studied. However, the exact mechanisms by which these pathogens cause disease remain unknown. Citrobacter rodentium (CR) is a well described murine bacterial pathogen and CR infection is a robust, relevant In Vivo model that can be used to understand the pathogenicity of EPEC and EHEC induced colitis. It is well known that MMP9 is absent in normal tissues but upregulated during inflammation. Aim of the present study was to identify the role played by MMP9 in CR-induced colitis. Methods: Age and gender matched MMP9-/- and littermate wild type (WT) mice of C57/B6 strain were used for the study. One group, including both MMP9-/and WT animals, were infected with CR and another group, without infection served as a control. Half of the animals in each group were sacrificed at days 8 and 20 post-infection respectively. Colon tissue was subjected to bacterial colony counts, histological staining, Western blotting and Q-PCR. Results: CR colony count was significantly lower in MMP9-/mice compared to WT mice (5.81±0.93x103 CFU vs. 23.78±7.2x103 CFU/g, respectively) on day 8 post-infection. Infected MMP9-/- mice also showed a significantly lower colon weight (0.05±0.005 g) compared to WT mice (0.07±0.005 g). MMP9 protein expression was increased by 5±0.2-fold at day 8 post-infection with CR in WT mice compared to uninfected WT mice. Muc2 expression was significantly lower (48.9±12.9%) in MMP9-/mice infected with CR compared to uninfected MMP9-/- mice on day 8 post-infection. Hematoxylin & Eosin staining revealed significantly decreased neutrophils infiltration, less crypt damage and fewer foci of ulceration in the colonic epithelium of MMP9-/- mice compared to WT mice both infected with CR on day 8 post-infection. On day 20 postinfection, crypt architecture was restored as well as neutrophil infiltration was less indicating the recovery from ulceration in both MMP9-/- and WT mice. However, the extent of epithelial dysplasia was greater in MMP9-/- mice compared to WT animals. Ki67 staining indicated higher colonic epithelial cell proliferation among MMP9-/- mice compared to WT mice on day 20 post-infection. Thus, at early stages of CR-mediated colitis, MMP9-/- mice were
AGA Abstracts
Mo2050 Suppression of MLK3 Signaling Selectively Modulates MAPK and PTEN Signaling During Intestinal Mucosal Cell Migration Pavlo L. Kovalenko, Lyudmyla Kunovska, Jian Chen, Kathleen A. Gallo, Marc D. Basson Mixed-lineage kinase-3 (MLK3) variably influences diverse MAPK pathways that may in turn modulate migration and proliferation in various cell types In Vitro. In a previous preliminary study, we observed that loss of MLK3 expression reduces intestinal epithelial mucosal healing In Vivo after inducing ulcers by serosal acetic acid In Vivo and MLK3 inhibition reduced Caco-2 ERK and JNK signaling In Vitro. We now sought to evaluate other potential downstream consequences of MLK3 loss, including MAPK and PTEN signaling, proliferation, and tissue morphology, as all these parameters might be relevant to mucosal wound healing. We compared jejunums from MLK3 knockout and wild type mice In Vivo and assessed the role of MLK3 signaling in human Caco-2 intestinal epithelial cells In Vitro after MLK3 inhibition. The MLK3 knockout mice exhibited significantly greater thickness
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which is a glucosinolate precursor of SFN, was orally administered to the mice, at dose of 8.5 mg/kg, before and after the injection of IND. SGS is converted to biologically active SFN in the intestinal lumen by myrosinase activity in the intestinal microflora. Expression of hemoxygenase-1 (HO-1) was evaluated by real time RT-PCR. Vascular permeability was assessed by Evans Blue exudation into the mucosa after i.v. injection. Neutrophil activation was evaluated by myeloperoxidase (MPO) activity. Results: 1. IND treatment caused mucosal injury in small intestine, accompanied by the increases in the vascular permeability, the mucosal MPO activity, and the mucosal invasion of anaerobic enterobacteria. 2.SGS attenuated the IND-induced small intestinal injury, and prevented the increases in the vascular permeability and the MPO activity induced by IND. 3. SGS enhanced HO-1 expression in the small intestine, and prevented the increase in mucosal invasion of the anaerobic enterobacteria caused by IND. Conclusions: These results suggest that (1) orally administered SGS protects small intestine from IND-induced injury in mice In Vivo, and that (2) SGS affords protection of the small intestinal mucosa against IND-induced injury, partly by inducing nrf2-dependent antioxidant enzymes in the mucosa, and partly by inhibiting invasion of the anaerobic enterobacteria into the mucosa.
resistant to inflammation, but at later stage exhibited increased dysplasia and proliferation, suggesting its dual role in the course of the disease. Conclusion: The data suggest that the MMP-9 initially mediates inflammation but later exerts a protective role, implying that function of MMP-9 changes as inflammation advances in CR-mediated colitis. Mo2049 Repeated Cycles of DSS Inducing a Chronically Relapsing Inflammation: A Novel Model to Study Fibrosis Using In Vivo MRI T2 Relaxometry Christine M. Breynaert, Tom Dresselaers, Jonathan Cremer, Kristel Van Steen, Clémentine Perrier, Severine Vermeire, Paul J. Rutgeerts, Jan L. Ceuppens, Uwe Himmelreich, Gert Van Assche BACKGROUND Most experimental animal models of IBD fail to accurately reflect the chronically relapsing inflammation underlying the complications of human Crohn's disease. This study investigated whether repeated cycles of DSS adequately reflect the effects of chronic transmural healing. Transmural μMR imaging with In Vivo T2 relaxometry was used to differentiate transmural changes. METHODS DSS colitis was induced in 6 week-old C57BL6/J mice: acute colitis mice (n=10) received 7 days of DSS prior to sacrifice. One cycle mice (n=10) received 1 cycle of 7 days of DSS followed by 2 weeks of normal drinking water prior to sacrifice, 2-cycle mice (n=10) 2 cycles and 3-cycle mice (n=9) 3 cycles prior to sacrifice. Control mice (n=7) received normal drinking water only. Six mice per group were scanned In Vivo on a 9.4T MRI Bruker system. T2 weighted images and T2 maps of the distal colon were recorded. Histograms of the colon wall were created from T2 maps using a plugin for ImageJ. After scanning and euthanasia, the distal colon of all mice was harvested for histology and FACS analysis. Collagen deposition was quantified with MartiusScarlett-Blue staining. RESULTS Colon weight, colon weight/length ratio and macroscopic score were significantly higher in the 1-, 2- and 3-cycle group compared to the control group (p<0.001), however, no significant difference was observed within the cycling groups. Although all mice in the 2-, and 3-cycle group had a normal DAI score, the macroscopic score was significantly higher compared to the acute group (p<0.001). CD4+Foxp3+ cells in blood increased with more cycles of DSS (p<0.001). The number of CD4+Foxp3+ cells in MLN was higher in the 2- and 3-cycle model compared to the acute model and controls (p<0.001). A higher number of IFNg+ cells in MLN was observed in the 1-, 2- and 3-cycle model compared to the acute model p<0.001). The 3-cycle model showed a higher number of IL17+ cells in MLN compared to the acute model and controls (p=0.0012). No significant difference was observed within the different models in IL13. Collagen deposition was significantly higher in the 2- and 3-cycle model compared to the 1-cycle model (p<0.001). T2 mapping of the colon was able to discern between all groups: the increasing number of cycles correlated with a gradual regression of T2 values to those of normal colon. CONCLUSION The immune profile of a cycling DSS model with induction of relapse and remission is clearly different from the acute DSS model inducing an adaptive immune response. The chronic repeated cycles of DSS model opens perspectives to study the effects of healing and fibrosis in a murine model of IBD. In Vivo T2 relaxometry is a promising non-invasive assessment of inflammation and fibrosis and should be explored in CD patients.
Mo2047 Hydrogen Sulfide Synthesis During Colonic Inflammation Occurs Primarily via a Pyridoxal-5'-Phosphate (P5p)-Independent Pathway Kyle L. Flannigan, John L. Wallace Hydrogen sulfide (H2S) is an important endogenous anti-inflammatory mediator, which has been shown to promote resolution of colitis and healing of ulcers. H2S synthesis was believed to occur primarily via two P5P-dependent enzymes (cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE)). In the present study, we examined the possible contribution of a third pathway for H2S synthesis, in the healthy and inflamed colon, which involves the enzymes cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3MST). Methods: The capacity of tissue to produce H2S was measured from homogenized tissue in the presence of exogenous substrate and/or inhibitors using a zinc-trapping assay. Production of H2S via the CAT-3MST pathway was explored using the substrate α-ketoglutarate and the CAT inhibitors L-aspartate and O-carboxymethyl-hydroxylamine hemihydrochloride (CHH). Colitis was induced in rats by intracolonic administration of dinitrobenzene sulfonic acid (DNBS). Rats were euthanized 3 to 28 days post-DNBS for measurement of H2S synthesis, and for immunofluorescent staining to localize CAT and 3MST expression. Results: Colonic H2S synthesis via the CAT-3MST pathway was 5-fold greater than that via the P5P-dependent (CBS and CSE) pathways (225 ± 59 vs. 42 ± 21 nmol/g/h respectively; p<0.05). In the inflamed colon, H2S synthesis via CAT-3MST was markedly increased (>4-fold) compared to controls (964 ± 115 vs. 225 ± 59 nmol/g/h, respectively; p<0.05). Colonic H2S synthesis was greatest when inflammation was most robust (3 days post-DNBS; 1132 ± 88 nmol/g/ h) and was suppressed by L-aspartate and CHH (424 ± 153 and 69 ± 34 nmol/g/h, respectively; both p<0.005). Immunofluorescence staining revealed that CAT and 3MST were colocalized in the colonic epithelium. Moreover, CAT expression was greatest in epithelial cells adjacent to ulcers (MFI: 1202 ± 63 U vs. 873 ± 105 U in healthy epithelium; p<0.05). By day 28, when colitis had largely resolved, H2S synthesis had decreased to control levels. Conclusions: These data suggest that in both the healthy and inflamed colon CAT-3MST represents the primary pathway for H2S synthesis. Increased CAT expression may account for the increased H2S synthesis during inflammation. Given the ability of H2S to promote resolution of inflammation and healing of mucosal injury, understanding the regulation of intestinal H2S synthesis may have important implications for the development of novel therapies for inflammatory bowel disease. Mo2048 Dual Roles Played by Matrix Metalloproteinase (MMP9) in Citrobacter rodentium-induced Hemorrhagic Colitis Sabrina Jeppsson, Maiko Sasaki, Jan-Michael A. Klapproth, Christopher W. Harper, Didier Merlin, Shanthi V. Sitaraman, Pallavi Garg Background and Aims: Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are well known causative agents of diarrheal disease in humans. EPEC is responsible for infantile diarrhea whereas EHEC causes hemorrhagic colitis. The molecular pathogenesis of EPEC and EHEC related infections have been well studied. However, the exact mechanisms by which these pathogens cause disease remain unknown. Citrobacter rodentium (CR) is a well described murine bacterial pathogen and CR infection is a robust, relevant In Vivo model that can be used to understand the pathogenicity of EPEC and EHEC induced colitis. It is well known that MMP9 is absent in normal tissues but upregulated during inflammation. Aim of the present study was to identify the role played by MMP9 in CR-induced colitis. Methods: Age and gender matched MMP9-/- and littermate wild type (WT) mice of C57/B6 strain were used for the study. One group, including both MMP9-/and WT animals, were infected with CR and another group, without infection served as a control. Half of the animals in each group were sacrificed at days 8 and 20 post-infection respectively. Colon tissue was subjected to bacterial colony counts, histological staining, Western blotting and Q-PCR. Results: CR colony count was significantly lower in MMP9-/mice compared to WT mice (5.81±0.93x103 CFU vs. 23.78±7.2x103 CFU/g, respectively) on day 8 post-infection. Infected MMP9-/- mice also showed a significantly lower colon weight (0.05±0.005 g) compared to WT mice (0.07±0.005 g). MMP9 protein expression was increased by 5±0.2-fold at day 8 post-infection with CR in WT mice compared to uninfected WT mice. Muc2 expression was significantly lower (48.9±12.9%) in MMP9-/mice infected with CR compared to uninfected MMP9-/- mice on day 8 post-infection. Hematoxylin & Eosin staining revealed significantly decreased neutrophils infiltration, less crypt damage and fewer foci of ulceration in the colonic epithelium of MMP9-/- mice compared to WT mice both infected with CR on day 8 post-infection. On day 20 postinfection, crypt architecture was restored as well as neutrophil infiltration was less indicating the recovery from ulceration in both MMP9-/- and WT mice. However, the extent of epithelial dysplasia was greater in MMP9-/- mice compared to WT animals. Ki67 staining indicated higher colonic epithelial cell proliferation among MMP9-/- mice compared to WT mice on day 20 post-infection. Thus, at early stages of CR-mediated colitis, MMP9-/- mice were
AGA Abstracts
Mo2050 Suppression of MLK3 Signaling Selectively Modulates MAPK and PTEN Signaling During Intestinal Mucosal Cell Migration Pavlo L. Kovalenko, Lyudmyla Kunovska, Jian Chen, Kathleen A. Gallo, Marc D. Basson Mixed-lineage kinase-3 (MLK3) variably influences diverse MAPK pathways that may in turn modulate migration and proliferation in various cell types In Vitro. In a previous preliminary study, we observed that loss of MLK3 expression reduces intestinal epithelial mucosal healing In Vivo after inducing ulcers by serosal acetic acid In Vivo and MLK3 inhibition reduced Caco-2 ERK and JNK signaling In Vitro. We now sought to evaluate other potential downstream consequences of MLK3 loss, including MAPK and PTEN signaling, proliferation, and tissue morphology, as all these parameters might be relevant to mucosal wound healing. We compared jejunums from MLK3 knockout and wild type mice In Vivo and assessed the role of MLK3 signaling in human Caco-2 intestinal epithelial cells In Vitro after MLK3 inhibition. The MLK3 knockout mice exhibited significantly greater thickness
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