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Su1869 Submucosal Fibrosis in Ulcerative Colitis Is Linked With Severity and Chronicity of Inflammation
Su1870 Differing Microbial Populations Induce TL1A-Mediated Intestinal Fibrosis Independently of TL1A-Mediated Inflammation Noam Jacob, Kotaro Kumagai, Yoshitake Kanazawa, Ariel M. Hamill, Erica Flores, YoungHee Kim, R. Balfour Sartor, Stephan Targan, Jonathan Jacobs, David Q. Shih
Comparison in rate of malignancy between IBD patients who have had Infliximab and htose who have not. There was no evidence that the adminstration of Inflidiman affected the rate of cancer amongst the IBD population.
Background: TL1A is an IBD associated gene that modulates the location and severity of intestinal inflammation and fibrosis. In IBD, TL1A is elevated in gut mucosa, characterized by fibrostenosis and need for surgery. Tl1a overexpression in mice causes ileitis, and worsens proximal colitis and fibrosis during colitogenic conditions. Differing microbial populations of the gut microbiome are associated with inflammatory disorders including IBD. The potential effect of these populations and their interaction with TL1A on mucosal fibrosis has not been investigated. Methods: Tl1a-Tg (sustained Tl1a expression) mice were rederived into germ-free (GF) status at the National Gnotobiotic Resource Center. 8wk-old, littermate WT or Tl1a-Tg mice were gavaged with either Cedars-Sinai specific pathogen free (SPF) stool or Human stool from a healthy human donor. Mice over 10mo of age were analyzed for intestinal fibrosis and inflammation. Picrosirius red stained sections were quantitated for amount of fibrosis. Intestinal inflammation was evaluated by H&E histological analyses. Results: We compared WT and Tl1a-Tg mice with respect to histologic inflammation and fibrosis under different microflora conditions (Table 1). Although there were increased spontaneous ileitis and collagen deposition in the cecum of Tl1a-Tg mice as compared to WT mice when raised with native SPF microbiome, these differences are abrogated under GF, or GF conditions reconstituted with SPF or human stool flora. However, the abrogation of ileitis between WT and Tg mice appears to be due to increased ileal inflammation seen in WT mice under GF or flora-reconstituted GF conditions. Thus, different microflora conditions may alter phenotypic expression of genotype-associated intestinal inflammation and fibrosis. To address this potential effect, we further compared mice raised under native SPF to those in GF conditions. GF WT mice developed increased ileitis and colitis as compared to WT mice raised with native SPF. Although there were no differences in intestinal inflammation in Tg mice with or w/o microflora, the absence of flora significantly reduced collagen deposition (Table 2). This suggests that commensal flora is required for Tl1a-mediated intestinal fibrosis. We next assessed if different microflora alter Tl1a-mediated inflammation and fibrosis by reconstitution with SPF or human flora. Different microflora do not alter intestinal inflammation and fibrosis in WT mice. However, Tl1a-mediated spontaneous ileitis and increased collagen deposition is associated with Cedars SPF, but not with human flora (Table 2). These data indicate that Tl1a-mediated intestinal inflammation and fibrosis depends on specific commensal flora composition. Conclusion: Tl1a-mediated intestinal fibrosis is independent of intestinal inflammation, but dependent on specific commensal flora composition seen in Cedars SPF. Table 1
Su1868 Dissecting the Role of RAGE in Intestinal Fibrosis Silvia Speca, Mathilde Body-Malapel, Chantal Fradin, Madjid Djouina, Eric Boulanger, Ann Marie Schmidt, Pierre Desreumaux, Cecile Vignal Background: The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin super-family able to regulate chronic inflammation. The prolonged AGE exposition in kidney, liver and lung is related to an upregulation of alphaSMA, the main marker of myofibroblasts activation, resulting in an accumulation of extracellular matrix components (ECM) and consequent fibrosis. At intestinal level fibrosis is a common and severe compliance of inflammatory bowel disease (IBD) and inflamed colonic mucosa of patients with active IBD shows a significant increase of AGE and RAGE. Our aim was to determine the role of RAGE in intestinal fibrogenesis. Methods: Fibrosis was induced in C57BL/6 wild type (WT) and RAGE-/- mice by for three cycles of 2,5% (w/v) dextran sulfate sodium (DSS) administration for six weeks, thus the entire colon was excised and scored for the assessment of macroscopic lesions (including dilation, thickness and adhesion) on a 0-3 scale. Distal colon specimens were subject to Hematoxylin/Eosin staining and Picrosirius red staining, to assess inflammation and collagen deposition. mRNA expression of the main profibrotic mediator, TGF-beta (Tgf-beta1 gene), alphaSMA (ACTA-1gene) and the expression of ECM components, mainly collagen types I-III ( Col1A1 gene) and fibronectin (FN-1 gene), were evaluated by quantitative RT-PCR. RAGE gene expression levels were also evaluated in TGF-beta-stimulated intestinal fibroblasts and epithelial cells, as well as in human primary intestinal fibroblasts isolated from IBD patients. Main Results: Compared to WT mice, DSStreated C57/Bl6 RAGE-/- mice showed a significant decrease of the colon weight/length ratio (29%, p< 0.0001), an indicator of wall thickening. In RAGE-/-mice, the macroscopic score was significantly reduced compared to WT mice (6 ± 0.92 vs 1.43 ± 0.54, p< 0.0001). DSS-treated RAGE-/- mice showed also a significant decrease of total microscopic score compared to WT mice (49%, p< 0.001). DSS administration also induced significant increase of Tgf-beta1, ACTA-1, Col1A1 and FN-1 gene expression in WT mice colon. DSS-induced upregulation of these genes was regulated in RAGE-/- mice for Col1A1 (3.17 fold, p< 0.01), Tgf-beta1 (2 fold, p< 0.01), ACTA-1 (1.2 fold, p< 0.05)and totally prevented for FN-1. In addition, myofibroblasts deriving by TGF-beta-treated intestinal fibroblasts and epithelial cells showed enhanced mRNA RAGE expression by 1.5 fold (p< 0.01) and 2.3 fold (p< 0.01), respectively. Also in primary human intestinal fibroblasts, obtained by UC patients, a significant increase by 3.8 fold (p < 0.05) of RAGE gene expression was observed. Conclusions: This study represents a first insight of the potential profibrotic role of RAGE in the development of intestinal fibrosis, shedding light into the complex and dynamic fibrogenic IBD.
Su1869 Submucosal Fibrosis in Ulcerative Colitis Is Linked With Severity and Chronicity of Inflammation Neha Agrawal, Eric Willis, Rocio Lopez, Bret Lashner, Claudio Fiocchi, Ilyssa Gordon, Florian Rieder Mean ± SD Table 2
Background: Chronic intestinal inflammation and impaired tissue repair leading to intestinal fibrosis is a well-recognized complication in Crohn's disease (CD), while the development of fibrosis in ulcerative colitis (UC) has remained largely unexplored. We aimed at characterizing the location and severity of UC associated fibrosis and its link with clinical parameters. Methods: 753 individual H&E stained tissue cross sections derived every 10 cm in 94 consecutive UC colectomy specimens were examined. Crohn's colitis (CD), diverticulitis (DIV) and healthy margins from colorectal cancer patients served as controls. Degree of inflammation (Geboes score), degree of fibrosis and morphometric measurements of all layers of the intestinal wall were determined. Four GI pathologists independently assessed representative sections stained with Masson Trichrome (MT) and H&E for presence and degree of fibrosis. Clinical data were collected. Results: 53.2% of the subjects were male. The median disease duration at time of colectomy was 64.3 months. At time of colectomy 89.4% had refractory disease and 94.7% had extensive colitis. Submucosal (SM) fibrosis was detected in 100% of UC colectomy specimens but only in areas of the colon affected by inflammation. Inflammation was associated with a thickening of the colonic wall, due to increased thickness of the lamina propria (LP) and muscularis mucosae (MM), but not the other tissue layers, including the SM (p<0.001). At the time of colectomy SM fibrosis was associated with histopathologic changes of chronic, but not active inflammation. The severity of inflammation was linked to the degree of SM fibrosis (Spearman correlations rho
Mean ± SD
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(95% confidence interval): 0.58 (0.41, 0.75); p<0.001). Male gender, refractory disease, steroids or 5-ASA at time of colectomy, but not disease extent or smoking were associated with increased degree of SM fibrosis (p=0.022 - 0.007). The use of 5-ASA and patient age at time of colectomy were associated with an increased thickness of the MM (p=0.043 0.005). The MM thickness and degree of SM fibrosis was not different between CD, UC and DIV. Inter-rater reliability was kappa 0.57 (for MT) and 0.63 (for H&E) and inter-rater agreement for degree of fibrosis between MT and H&E was kappa 0.62 - 0.69. Conclusions: Severity and histopathologic signs of chronic inflammation are linked with SM fibrosis and increased wall thickness in UC. Male gender, refractory disease as well as steroids or 5-ASA use at time of colectomy are associated with the degree of fibrotic burden and 5-ASA and patient age with thickness of the MM. While this cross sectional study does not allow a cause and effect conclusion, UC should be considered a progressive disease with a significant degree of fibrosis and wall thickening, possibly leading to complications such as motility abnormalities and increased wall stiffness.
Comparison in rate of malignancy between IBD patients who have had Infliximab and htose who have not. There was no evidence that the adminstration of Inflidiman affected the rate of cancer amongst the IBD population.
Background: TL1A is an IBD associated gene that modulates the location and severity of intestinal inflammation and fibrosis. In IBD, TL1A is elevated in gut mucosa, characterized by fibrostenosis and need for surgery. Tl1a overexpression in mice causes ileitis, and worsens proximal colitis and fibrosis during colitogenic conditions. Differing microbial populations of the gut microbiome are associated with inflammatory disorders including IBD. The potential effect of these populations and their interaction with TL1A on mucosal fibrosis has not been investigated. Methods: Tl1a-Tg (sustained Tl1a expression) mice were rederived into germ-free (GF) status at the National Gnotobiotic Resource Center. 8wk-old, littermate WT or Tl1a-Tg mice were gavaged with either Cedars-Sinai specific pathogen free (SPF) stool or Human stool from a healthy human donor. Mice over 10mo of age were analyzed for intestinal fibrosis and inflammation. Picrosirius red stained sections were quantitated for amount of fibrosis. Intestinal inflammation was evaluated by H&E histological analyses. Results: We compared WT and Tl1a-Tg mice with respect to histologic inflammation and fibrosis under different microflora conditions (Table 1). Although there were increased spontaneous ileitis and collagen deposition in the cecum of Tl1a-Tg mice as compared to WT mice when raised with native SPF microbiome, these differences are abrogated under GF, or GF conditions reconstituted with SPF or human stool flora. However, the abrogation of ileitis between WT and Tg mice appears to be due to increased ileal inflammation seen in WT mice under GF or flora-reconstituted GF conditions. Thus, different microflora conditions may alter phenotypic expression of genotype-associated intestinal inflammation and fibrosis. To address this potential effect, we further compared mice raised under native SPF to those in GF conditions. GF WT mice developed increased ileitis and colitis as compared to WT mice raised with native SPF. Although there were no differences in intestinal inflammation in Tg mice with or w/o microflora, the absence of flora significantly reduced collagen deposition (Table 2). This suggests that commensal flora is required for Tl1a-mediated intestinal fibrosis. We next assessed if different microflora alter Tl1a-mediated inflammation and fibrosis by reconstitution with SPF or human flora. Different microflora do not alter intestinal inflammation and fibrosis in WT mice. However, Tl1a-mediated spontaneous ileitis and increased collagen deposition is associated with Cedars SPF, but not with human flora (Table 2). These data indicate that Tl1a-mediated intestinal inflammation and fibrosis depends on specific commensal flora composition. Conclusion: Tl1a-mediated intestinal fibrosis is independent of intestinal inflammation, but dependent on specific commensal flora composition seen in Cedars SPF. Table 1
Su1868 Dissecting the Role of RAGE in Intestinal Fibrosis Silvia Speca, Mathilde Body-Malapel, Chantal Fradin, Madjid Djouina, Eric Boulanger, Ann Marie Schmidt, Pierre Desreumaux, Cecile Vignal Background: The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin super-family able to regulate chronic inflammation. The prolonged AGE exposition in kidney, liver and lung is related to an upregulation of alphaSMA, the main marker of myofibroblasts activation, resulting in an accumulation of extracellular matrix components (ECM) and consequent fibrosis. At intestinal level fibrosis is a common and severe compliance of inflammatory bowel disease (IBD) and inflamed colonic mucosa of patients with active IBD shows a significant increase of AGE and RAGE. Our aim was to determine the role of RAGE in intestinal fibrogenesis. Methods: Fibrosis was induced in C57BL/6 wild type (WT) and RAGE-/- mice by for three cycles of 2,5% (w/v) dextran sulfate sodium (DSS) administration for six weeks, thus the entire colon was excised and scored for the assessment of macroscopic lesions (including dilation, thickness and adhesion) on a 0-3 scale. Distal colon specimens were subject to Hematoxylin/Eosin staining and Picrosirius red staining, to assess inflammation and collagen deposition. mRNA expression of the main profibrotic mediator, TGF-beta (Tgf-beta1 gene), alphaSMA (ACTA-1gene) and the expression of ECM components, mainly collagen types I-III ( Col1A1 gene) and fibronectin (FN-1 gene), were evaluated by quantitative RT-PCR. RAGE gene expression levels were also evaluated in TGF-beta-stimulated intestinal fibroblasts and epithelial cells, as well as in human primary intestinal fibroblasts isolated from IBD patients. Main Results: Compared to WT mice, DSStreated C57/Bl6 RAGE-/- mice showed a significant decrease of the colon weight/length ratio (29%, p< 0.0001), an indicator of wall thickening. In RAGE-/-mice, the macroscopic score was significantly reduced compared to WT mice (6 ± 0.92 vs 1.43 ± 0.54, p< 0.0001). DSS-treated RAGE-/- mice showed also a significant decrease of total microscopic score compared to WT mice (49%, p< 0.001). DSS administration also induced significant increase of Tgf-beta1, ACTA-1, Col1A1 and FN-1 gene expression in WT mice colon. DSS-induced upregulation of these genes was regulated in RAGE-/- mice for Col1A1 (3.17 fold, p< 0.01), Tgf-beta1 (2 fold, p< 0.01), ACTA-1 (1.2 fold, p< 0.05)and totally prevented for FN-1. In addition, myofibroblasts deriving by TGF-beta-treated intestinal fibroblasts and epithelial cells showed enhanced mRNA RAGE expression by 1.5 fold (p< 0.01) and 2.3 fold (p< 0.01), respectively. Also in primary human intestinal fibroblasts, obtained by UC patients, a significant increase by 3.8 fold (p < 0.05) of RAGE gene expression was observed. Conclusions: This study represents a first insight of the potential profibrotic role of RAGE in the development of intestinal fibrosis, shedding light into the complex and dynamic fibrogenic IBD.
Su1869 Submucosal Fibrosis in Ulcerative Colitis Is Linked With Severity and Chronicity of Inflammation Neha Agrawal, Eric Willis, Rocio Lopez, Bret Lashner, Claudio Fiocchi, Ilyssa Gordon, Florian Rieder Mean ± SD Table 2
Background: Chronic intestinal inflammation and impaired tissue repair leading to intestinal fibrosis is a well-recognized complication in Crohn's disease (CD), while the development of fibrosis in ulcerative colitis (UC) has remained largely unexplored. We aimed at characterizing the location and severity of UC associated fibrosis and its link with clinical parameters. Methods: 753 individual H&E stained tissue cross sections derived every 10 cm in 94 consecutive UC colectomy specimens were examined. Crohn's colitis (CD), diverticulitis (DIV) and healthy margins from colorectal cancer patients served as controls. Degree of inflammation (Geboes score), degree of fibrosis and morphometric measurements of all layers of the intestinal wall were determined. Four GI pathologists independently assessed representative sections stained with Masson Trichrome (MT) and H&E for presence and degree of fibrosis. Clinical data were collected. Results: 53.2% of the subjects were male. The median disease duration at time of colectomy was 64.3 months. At time of colectomy 89.4% had refractory disease and 94.7% had extensive colitis. Submucosal (SM) fibrosis was detected in 100% of UC colectomy specimens but only in areas of the colon affected by inflammation. Inflammation was associated with a thickening of the colonic wall, due to increased thickness of the lamina propria (LP) and muscularis mucosae (MM), but not the other tissue layers, including the SM (p<0.001). At the time of colectomy SM fibrosis was associated with histopathologic changes of chronic, but not active inflammation. The severity of inflammation was linked to the degree of SM fibrosis (Spearman correlations rho
Mean ± SD
S-575
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Page 575
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(95% confidence interval): 0.58 (0.41, 0.75); p<0.001). Male gender, refractory disease, steroids or 5-ASA at time of colectomy, but not disease extent or smoking were associated with increased degree of SM fibrosis (p=0.022 - 0.007). The use of 5-ASA and patient age at time of colectomy were associated with an increased thickness of the MM (p=0.043 0.005). The MM thickness and degree of SM fibrosis was not different between CD, UC and DIV. Inter-rater reliability was kappa 0.57 (for MT) and 0.63 (for H&E) and inter-rater agreement for degree of fibrosis between MT and H&E was kappa 0.62 - 0.69. Conclusions: Severity and histopathologic signs of chronic inflammation are linked with SM fibrosis and increased wall thickness in UC. Male gender, refractory disease as well as steroids or 5-ASA use at time of colectomy are associated with the degree of fibrotic burden and 5-ASA and patient age with thickness of the MM. While this cross sectional study does not allow a cause and effect conclusion, UC should be considered a progressive disease with a significant degree of fibrosis and wall thickening, possibly leading to complications such as motility abnormalities and increased wall stiffness.