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W1847 Increased Matrix Stiffness Induces a Fibrogenic Phenotype in Human Colonic Mesenchymal Cells
AGA Abstracts
mechanisms underpinning the B. animalis AHC7 protective effect; (2) To determine if the immunological effect also applies to other bacterial strains. Materials & Methods. Healthy mice consumed B. animalis AHC7 for 3 weeks followed by isolation of Peyer's patches and mesenteric lymph nodes. Single cell suspensions were generated and analyzed In Vitro for cytokine production and superoxide dismutase (SOD) activity. Transgenic NF-κBlux mice were fed B. animalis AHC7 or Enterococcus faecium followed by deliberate infection with S. typhimurium. Whole body NF-κB activation was determined using biophotonic imaging. Results. In Vitro anti-CD3/CD28 stimulated Peyer's patch cells secreted significantly less TNF-α and IFN-γ following B. animalis AHC7 consumption. Stimulated cells released more IL-12p70 but this difference did not reach statistical significance. No alteration in IL-6, IL10, MCP-1 or SOD levels were observed. No change in the cytokine profile of mesenteric lymph node cells was noted. Following 4 hours of infection with S. typhimurium, NF-κB activation was significantly elevated In Vivo in placebo and E. faecium-fed animals while B. animalis AHC7 consumption completely blocked the NF-κB response. Conclusion. B. animalis AHC7 exerts a strain-specific anti-inflammatory effect via the inhibition of proinflammatory cytokine production and blockade of pro-inflammatory transcription factor activation in response to an infectious insult. These results suggest that this bacterium may have utility in the management of inflammatory diseases, in particular inflammation associated with infection.
W1847 Increased Matrix Stiffness Induces a Fibrogenic Phenotype in Human Colonic Mesenchymal Cells Luz P. Blanco, Laura Johnson, Kay L. Sauder, Florian Rieder, Claudio Fiocchi, Fei Liu, Justin Mih, Daniel Tschumperlin, Peter D. Higgins Background: Fibrotic strictures are one of the most serious complications of Crohn's disease. Despite current anti-inflammatory therapies, the majority of patients progress to stenosis and surgery. This progression suggests that fibrogenesis can become independent of inflammation, and auto-propagative. We hypothesized that colonic mesenchymal cells may be able to sense a high-stiffness extracellular matrix (as occurs in intestinal fibrosis) and respond to it by proliferating, acquiring a myofibroblast-like phenotype, and becoming more fibrogenic. This, in turn, can further increase tissue stiffness and thus create a positive feedback loop. Methods: To study matrix sensing In Vitro we employed plates containing a stiffness gradient of polyacrylamide-filled wells coated with collagen. The polyacrylamide matrices varied from a shear modulus of 100 Pascals (Pa) to up to 1600 Pa and cells grown on these wells were compared to cells grown on glass wells (70 GPa). We tested the human colonic fibroblast cell line CCD-18Co and primary human mesenchymal colonic cells isolated from control and Crohn's disease patients. Results: The CCD-18Co fibroblast cell line, and primary mesenchymal cells from control or IBD colons proliferate at 2.71 to 6.26 fold higher rates on higher stiffness media (800 Pa and 1600 Pa) compared to 100 Pa (p< 0.01) after 6 days of culture. Between 400 Pa and 800 Pa there is a drastic change in the morphology of the cells from rounded to myofibroblast-like, and cell viability (measured using the CellTiter 96 AQueous One assay, Promega) is increased 1.64 fold at 800 Pa compared to 400 Pa (p< 0.02). With increasing stiffness (glass surface) the primary colonic cells acquire a resistance to apoptosis with 5.39-5.95 fold less apoptosis compared to 100 Pa (p< 0.05) measured using the Apo-One caspase-3/7 assay from Promega. By Q-RT-PCR the matrix metalloproteinases MMP-1 and MMP-10 are down-regulated 7.85 and 48.39 fold at the RNA level, at 1600 Pa vs. 100 Pa in CCD-18Co cells. In contrast, endothelin-1 gene expression is induced 2.81 to 13.93 fold in the different cell types on 1600 Pa versus 100 Pa matrices. Conclusions: Human primary colonic cells and fibroblasts cells are able to sense matrix stiffness, resulting in changes that affect cellular morphology, cell replication, apoptosis sensitivity, MMPs and endothelin-1 gene expression. Our results support the hypothesis that high stiffness matrices (≥ 800 Pa) can induce a fibrogenic phenotype in human intestinal mesenchymal cells which may lead to a positive feedback loop and auto-propagation of intestinal fibrosis.
W1845 Longitudinal Analysis of Bacteroidales in the Human Intestinal Ecosystem Naamah Zitomersky, Michael Coyne, Laurie E. Comstock The intestinal microbiota is a diverse and thriving ecosystem estimated to contain bacteria at a concentration of approximately 1011-1012 per gram. Within the colonic ecosystem, Bacteroidales species are the most prevalent Gram negative bacteria. Although several studies have described large scale 16S rRNA gene-based sequencing analyses to determine the bacterial composition of human stool, few have described quantitative analyses to determine the levels of various species and how these levels may change over time within an individual's ecosystem. We performed a longitudinal culture-based analysis of total Bacteroidales concentrations and the prevalent Bacteroidales species in human stool samples from 15 healthy adults. Five samples were collected from each subject over a 12 month period. In all samples, Bacteroidales were present at 108-1011 cfu/gm of stool. Although the total levels of Bacteroidales remained somewhat constant within some individual's stool over time, log scale changes were detected in samples from some subjects. Prominent species were identified using a series of muliplex PCR assays that are based on the 16S rRNA gene region. These multiplex PCRs discriminate and identify 17 prevalent intestinal Bacteroidales species. The 16S rRNA gene was amplified and sequenced for those species which were not identified by the multiplex PCRs. The numbers of different Bacteroidales species that were present on the two highest dilution plates varied but were consistently greater than 3 and in some samples as many as 12 Bacteroidales species were identified at the level of 108. The predominant Bacteroidales species differed from subject to subject and in some cases, differed over time within an individual. The culture-based approach used in this study created a large strain collection. Using these strains and cataloged fecal samples and bacteriophage preparations obtained from each of these fecal samples, we are currently examining microbial and host factors that may have influenced some of the species-level changes that were observed within a subject's ecosystem over time.
W1848 A Pilot Study of Ultrasound Elasticity Imaging to Identify and Measure Segmental Intestinal Fibrosis in Patients With Crohn's Disease Ryan Stidham, Jingping Xu, Kang Kim, Jonathan M. Rubin, David Moons, Barbara J. McKenna, Sujal Rangwalla, Peter D. Higgins Background: Intestinal fibrosis in Crohn's disease results from cycles of inflammation and healing and is rarely identified prior to the onset of clinical symptoms. Methods of identifying and measuring intestinal fibrosis are needed. Ultrasound elasticity imaging (UEI) uses standard abdominal ultrasound (US) and novel methods of image analysis to determine the mechanical properties of tissues during deformation of the region of interest. Published data show that UEI differentiates fibrotic from normal intestine in rodent models, and is accurate in other human organs. Here we present the first human study of UEI as a diagnostic tool to differentiate normal from fibrotic bowel in patients with Crohn's disease. Aims: 1) To determine if UEI differentiates normal from abnormal bowel by strain measurements; and 2) To determine whether UEI can accurately predict the severity of fibrosis. Methods: Two subjects with Crohn's disease undergoing elective intestinal resection for stenosis, documented on prior cross-sectional imaging, underwent preoperative transcutaneous abdominal US exam. US data from ultrasonographically stenotic and normal-appearing bowel were captured and analyzed for strain using phase-sensitive two dimensional speckle tracking. UEI strain values were compared to both: (i) the mechanical properties of the surgical specimens with direct measurement using a custom ex vivo elastometer (Artann Labs, NJ) to calculate the Young's modulus; and (ii) histopathology. Results: In these two subjects, UEI consistently demonstrated less strain (less deformation during compression, or more stiffness) in the fibrotic bowel segments compared to normal bowel (see Table 1). This was consistent with ratios of Young's modulus provided by ex vivo elastometry. Intra-subject histopathologic comparison of normal and stenotic tissue revealed consistent increase in fibrosis based on Trichrome and H&E staining as well as architectural distortion and muscularis layer thickening in the stenotic tissue. Conclusions: This is the first application of UEI in human intestinal fibrosis. This appears to be a promising non-invasive method for assessing intestinal fibrosis when severe. Additional data continue to be collected in this ongoing study. Future studies are needed to determine the sensitivity and utility of UEI in mild fibrosis, the change in bowel stiffness over time in longitudinal studies, and the ability of UEI to distinguish inflammatory from fibrotic strictures.
W1846 Identification of Epithelial to Mesenchymal Transition as a Novel Source of Fibroblasts in Intestinal Fibrosis Harikrishna Tanjore, Sarah N. Flier, Efi Kokkotou, Hikaru Sugimoto, Michael Zeisberg, Raghu Kalluri Crohn's disease (CD) is a disorder of chronic, transmural inflammation that can affect any part of the gastrointestinal tract from the mouth to the anus. Chronic inflammation combined with dysregulated wound healing is thought to result in a number of complications including fistula and fibrostenotic stricture formation. Intestinal fibrosis in the form of strictures is a major complication of CD, but the precise mechanism by which it occurs is incompletely understood. As a result, specific therapies to halt or even reverse fibrosis have not been explored. Here, we evaluated the contribution of epithelial to mesenchymal transition (EMT) to intestinal fibrosis associated with a mouse model of CD. Mice administered intra-rectal 2,4,6-trinitrobenzene sulphonic acid (TNBS) develop inflammation and fibrosis that resembles CD both histologically and by immunologic profile. We utilized this mouse model to molecularly probe the contribution of EMT to intestinal fibrosis. Additionally, we utilized double-transgenic VillinCre;R26Rosa-lox-STOP-lox-LacZ mice, in which removal of the STOP cassette by Cre recombinase in villin-positive intestinal epithelial cells irreversibly activates permanent LacZ expression, to lineage trace epithelial cells that might undergo EMT upon TNBS administration. TNBS-induced fibrosis is associated with the presence of a significant number of cells which express both epithelial and mesenchymal markers. In the lineage tagged transgenic mice, appearance of LacZ-positive cells which also express the fibroblast marker FSP1 unequivocally demonstrates EMT. TGF-β1, a known inducer of EMT in epithelial cells, induces EMT in rat intestinal epithelial cells In Vitro and Bone Morphogenic Protein (BMP)-7, an antagonist of TGF-β1, inhibits EMT and fibrosis both In Vitro and in the TNBS-treated mice. Our study demonstrates that EMT contributes to intestinal fibrosis associated with the TNBS-induced model of Crohn's colitis and that inhibition of TGF-β1 with recombinant human BMP-7 prevents this process and prevents fibrosis.
AGA Abstracts
S-752
mechanisms underpinning the B. animalis AHC7 protective effect; (2) To determine if the immunological effect also applies to other bacterial strains. Materials & Methods. Healthy mice consumed B. animalis AHC7 for 3 weeks followed by isolation of Peyer's patches and mesenteric lymph nodes. Single cell suspensions were generated and analyzed In Vitro for cytokine production and superoxide dismutase (SOD) activity. Transgenic NF-κBlux mice were fed B. animalis AHC7 or Enterococcus faecium followed by deliberate infection with S. typhimurium. Whole body NF-κB activation was determined using biophotonic imaging. Results. In Vitro anti-CD3/CD28 stimulated Peyer's patch cells secreted significantly less TNF-α and IFN-γ following B. animalis AHC7 consumption. Stimulated cells released more IL-12p70 but this difference did not reach statistical significance. No alteration in IL-6, IL10, MCP-1 or SOD levels were observed. No change in the cytokine profile of mesenteric lymph node cells was noted. Following 4 hours of infection with S. typhimurium, NF-κB activation was significantly elevated In Vivo in placebo and E. faecium-fed animals while B. animalis AHC7 consumption completely blocked the NF-κB response. Conclusion. B. animalis AHC7 exerts a strain-specific anti-inflammatory effect via the inhibition of proinflammatory cytokine production and blockade of pro-inflammatory transcription factor activation in response to an infectious insult. These results suggest that this bacterium may have utility in the management of inflammatory diseases, in particular inflammation associated with infection.
W1847 Increased Matrix Stiffness Induces a Fibrogenic Phenotype in Human Colonic Mesenchymal Cells Luz P. Blanco, Laura Johnson, Kay L. Sauder, Florian Rieder, Claudio Fiocchi, Fei Liu, Justin Mih, Daniel Tschumperlin, Peter D. Higgins Background: Fibrotic strictures are one of the most serious complications of Crohn's disease. Despite current anti-inflammatory therapies, the majority of patients progress to stenosis and surgery. This progression suggests that fibrogenesis can become independent of inflammation, and auto-propagative. We hypothesized that colonic mesenchymal cells may be able to sense a high-stiffness extracellular matrix (as occurs in intestinal fibrosis) and respond to it by proliferating, acquiring a myofibroblast-like phenotype, and becoming more fibrogenic. This, in turn, can further increase tissue stiffness and thus create a positive feedback loop. Methods: To study matrix sensing In Vitro we employed plates containing a stiffness gradient of polyacrylamide-filled wells coated with collagen. The polyacrylamide matrices varied from a shear modulus of 100 Pascals (Pa) to up to 1600 Pa and cells grown on these wells were compared to cells grown on glass wells (70 GPa). We tested the human colonic fibroblast cell line CCD-18Co and primary human mesenchymal colonic cells isolated from control and Crohn's disease patients. Results: The CCD-18Co fibroblast cell line, and primary mesenchymal cells from control or IBD colons proliferate at 2.71 to 6.26 fold higher rates on higher stiffness media (800 Pa and 1600 Pa) compared to 100 Pa (p< 0.01) after 6 days of culture. Between 400 Pa and 800 Pa there is a drastic change in the morphology of the cells from rounded to myofibroblast-like, and cell viability (measured using the CellTiter 96 AQueous One assay, Promega) is increased 1.64 fold at 800 Pa compared to 400 Pa (p< 0.02). With increasing stiffness (glass surface) the primary colonic cells acquire a resistance to apoptosis with 5.39-5.95 fold less apoptosis compared to 100 Pa (p< 0.05) measured using the Apo-One caspase-3/7 assay from Promega. By Q-RT-PCR the matrix metalloproteinases MMP-1 and MMP-10 are down-regulated 7.85 and 48.39 fold at the RNA level, at 1600 Pa vs. 100 Pa in CCD-18Co cells. In contrast, endothelin-1 gene expression is induced 2.81 to 13.93 fold in the different cell types on 1600 Pa versus 100 Pa matrices. Conclusions: Human primary colonic cells and fibroblasts cells are able to sense matrix stiffness, resulting in changes that affect cellular morphology, cell replication, apoptosis sensitivity, MMPs and endothelin-1 gene expression. Our results support the hypothesis that high stiffness matrices (≥ 800 Pa) can induce a fibrogenic phenotype in human intestinal mesenchymal cells which may lead to a positive feedback loop and auto-propagation of intestinal fibrosis.
W1845 Longitudinal Analysis of Bacteroidales in the Human Intestinal Ecosystem Naamah Zitomersky, Michael Coyne, Laurie E. Comstock The intestinal microbiota is a diverse and thriving ecosystem estimated to contain bacteria at a concentration of approximately 1011-1012 per gram. Within the colonic ecosystem, Bacteroidales species are the most prevalent Gram negative bacteria. Although several studies have described large scale 16S rRNA gene-based sequencing analyses to determine the bacterial composition of human stool, few have described quantitative analyses to determine the levels of various species and how these levels may change over time within an individual's ecosystem. We performed a longitudinal culture-based analysis of total Bacteroidales concentrations and the prevalent Bacteroidales species in human stool samples from 15 healthy adults. Five samples were collected from each subject over a 12 month period. In all samples, Bacteroidales were present at 108-1011 cfu/gm of stool. Although the total levels of Bacteroidales remained somewhat constant within some individual's stool over time, log scale changes were detected in samples from some subjects. Prominent species were identified using a series of muliplex PCR assays that are based on the 16S rRNA gene region. These multiplex PCRs discriminate and identify 17 prevalent intestinal Bacteroidales species. The 16S rRNA gene was amplified and sequenced for those species which were not identified by the multiplex PCRs. The numbers of different Bacteroidales species that were present on the two highest dilution plates varied but were consistently greater than 3 and in some samples as many as 12 Bacteroidales species were identified at the level of 108. The predominant Bacteroidales species differed from subject to subject and in some cases, differed over time within an individual. The culture-based approach used in this study created a large strain collection. Using these strains and cataloged fecal samples and bacteriophage preparations obtained from each of these fecal samples, we are currently examining microbial and host factors that may have influenced some of the species-level changes that were observed within a subject's ecosystem over time.
W1848 A Pilot Study of Ultrasound Elasticity Imaging to Identify and Measure Segmental Intestinal Fibrosis in Patients With Crohn's Disease Ryan Stidham, Jingping Xu, Kang Kim, Jonathan M. Rubin, David Moons, Barbara J. McKenna, Sujal Rangwalla, Peter D. Higgins Background: Intestinal fibrosis in Crohn's disease results from cycles of inflammation and healing and is rarely identified prior to the onset of clinical symptoms. Methods of identifying and measuring intestinal fibrosis are needed. Ultrasound elasticity imaging (UEI) uses standard abdominal ultrasound (US) and novel methods of image analysis to determine the mechanical properties of tissues during deformation of the region of interest. Published data show that UEI differentiates fibrotic from normal intestine in rodent models, and is accurate in other human organs. Here we present the first human study of UEI as a diagnostic tool to differentiate normal from fibrotic bowel in patients with Crohn's disease. Aims: 1) To determine if UEI differentiates normal from abnormal bowel by strain measurements; and 2) To determine whether UEI can accurately predict the severity of fibrosis. Methods: Two subjects with Crohn's disease undergoing elective intestinal resection for stenosis, documented on prior cross-sectional imaging, underwent preoperative transcutaneous abdominal US exam. US data from ultrasonographically stenotic and normal-appearing bowel were captured and analyzed for strain using phase-sensitive two dimensional speckle tracking. UEI strain values were compared to both: (i) the mechanical properties of the surgical specimens with direct measurement using a custom ex vivo elastometer (Artann Labs, NJ) to calculate the Young's modulus; and (ii) histopathology. Results: In these two subjects, UEI consistently demonstrated less strain (less deformation during compression, or more stiffness) in the fibrotic bowel segments compared to normal bowel (see Table 1). This was consistent with ratios of Young's modulus provided by ex vivo elastometry. Intra-subject histopathologic comparison of normal and stenotic tissue revealed consistent increase in fibrosis based on Trichrome and H&E staining as well as architectural distortion and muscularis layer thickening in the stenotic tissue. Conclusions: This is the first application of UEI in human intestinal fibrosis. This appears to be a promising non-invasive method for assessing intestinal fibrosis when severe. Additional data continue to be collected in this ongoing study. Future studies are needed to determine the sensitivity and utility of UEI in mild fibrosis, the change in bowel stiffness over time in longitudinal studies, and the ability of UEI to distinguish inflammatory from fibrotic strictures.
W1846 Identification of Epithelial to Mesenchymal Transition as a Novel Source of Fibroblasts in Intestinal Fibrosis Harikrishna Tanjore, Sarah N. Flier, Efi Kokkotou, Hikaru Sugimoto, Michael Zeisberg, Raghu Kalluri Crohn's disease (CD) is a disorder of chronic, transmural inflammation that can affect any part of the gastrointestinal tract from the mouth to the anus. Chronic inflammation combined with dysregulated wound healing is thought to result in a number of complications including fistula and fibrostenotic stricture formation. Intestinal fibrosis in the form of strictures is a major complication of CD, but the precise mechanism by which it occurs is incompletely understood. As a result, specific therapies to halt or even reverse fibrosis have not been explored. Here, we evaluated the contribution of epithelial to mesenchymal transition (EMT) to intestinal fibrosis associated with a mouse model of CD. Mice administered intra-rectal 2,4,6-trinitrobenzene sulphonic acid (TNBS) develop inflammation and fibrosis that resembles CD both histologically and by immunologic profile. We utilized this mouse model to molecularly probe the contribution of EMT to intestinal fibrosis. Additionally, we utilized double-transgenic VillinCre;R26Rosa-lox-STOP-lox-LacZ mice, in which removal of the STOP cassette by Cre recombinase in villin-positive intestinal epithelial cells irreversibly activates permanent LacZ expression, to lineage trace epithelial cells that might undergo EMT upon TNBS administration. TNBS-induced fibrosis is associated with the presence of a significant number of cells which express both epithelial and mesenchymal markers. In the lineage tagged transgenic mice, appearance of LacZ-positive cells which also express the fibroblast marker FSP1 unequivocally demonstrates EMT. TGF-β1, a known inducer of EMT in epithelial cells, induces EMT in rat intestinal epithelial cells In Vitro and Bone Morphogenic Protein (BMP)-7, an antagonist of TGF-β1, inhibits EMT and fibrosis both In Vitro and in the TNBS-treated mice. Our study demonstrates that EMT contributes to intestinal fibrosis associated with the TNBS-induced model of Crohn's colitis and that inhibition of TGF-β1 with recombinant human BMP-7 prevents this process and prevents fibrosis.
AGA Abstracts
S-752