expression with time after transplantation was confirmed by real-time PCR and Sirius Red staining. Lumen obliteration was connected with increased expression of potent mediators of fibrosis such as TGF-β. CONCLUSION: We established a method for heterotopic transplantation of small bowel resections in mice. A variety of histologic and molecular features of fibrosis were observed in the heterotopic intestinal grafts which suggests, that this new in vivo model will be instrumental in studying pathogenesis and treatment of intestinal fibrosis. Mo1701 MYD88 Signaling Is Not Essential in Intestinal Fibrosis in Mice Christian Lutz, Michaela Caj, Michael Fried, Gerhard Rogler, Martin Hausmann BACKGROUND: Hallmark of fibrosis is an excessive deposition of collagenous extracellular matrix as a consequence of proliferation and activation of fibroblasts and myofibroblasts. A pivotal role of IL-1R1/MyD88 signaling was suggested in tissue-resident cells leading to lymphocyte recruitment and fibroblast activation. We investigated whether intestinal fibrosis occurs after heterotopic transplantation of small bowel resections lacking functional MyD88. METHODS: Donor (B6-MyD88) small bowel resections were transplanted subcutaneously into the neck of recipients (B6-Tg UBC-GFP) and vice versa. Grafts were explanted 1, 2, 3, 4, 5, 6, 7, 14 and 21 days after transplantation. RESULTS: Rapid loss of crypt structures at day one after intestinal transplantation was found in all grafts. Histologic cross sections were stained with EvG to highlight collagen. Freshly isolated samples exhibited very little collagen staining at the submucosal border (10.1 ± 2.3 μm). In contrast, collagen layer thickness was significantly increased at day seven after transplantation in allografts from both B6-MyD88 (20.8 ± 2.1 μm, p <0.001) and B6-Tg UBC-GFP (27.3 ± 10.1 μm, p <0.001). Increased collagen expression over time after transplantation was confirmed by Sirius Red staining and real-time PCR (1.1 ± 0.3 arbitrary units in freshly isolated small bowel as compared to day five 198.9 ± 89.4 for B6-MyD88 and 467.3 ± 178.9 for B6-Tg UBC-GFP grafts). Increased collagen was connected with increased expression of TGF-β (1.2 ± 0.4 arbitrary units in freshly isolated small bowel as compared to day five 4.0 ± 1.5 for B6MyD88 and 5.0 ± 3.7 for B6-Tg UBC-GFP grafts). CONCLUSION: Increased collagen and TGF-β were observed in the heterotopic intestinal grafts where B6-MyD88 were used either as a donor or recipient of small bowel resections. This suggests that MyD88 signaling is not essential for the induction of important profibrotic cytokine and formation of thickened collagen during the pathogenesis of intestinal fibrosis in mice.
Mo1699 Serum Metabolite and 454 Pyrosequencing Analysis Reveal the Presence of Oxidative Stress, Fecal Dysbiosis, and Dysfunction of the Fecal Microbiota in Spontaneous Canine Idiopathic Inflammatory Bowel Disease Yasushi Minamoto, Cristiane C. Otoni, Olga Büyükleblebici, Joerg M. Steiner, Albert Jergens, Jan S. Suchodolski Background: Idiopathic inflammatory bowel disease (IBD) is a common cause for chronic enteropathy in dogs. Similarly to human IBD, a combination of an altered GI microbiota (i.e., intestinal dysbiosis), an underlying host genetic susceptibility, and dietary and/or environmental factors are incriminated in the pathogenesis of canine IBD. Because intestinal inflammation develops spontaneously in the dog and the animals live in home environments, they may serve as a useful spontaneous model to study therapeutic approaches to IBD. Aim: To evaluate the fecal microbiota and serum metabolites in dogs with IBD. We also aimed to evaluate the effect of 21 days of medical therapy (immunosuppressive therapy and elimination diet) on these same profiles. Animals: 12 dogs with IBD showing clinical improvement after 21 days of medical therapy and 10 healthy control dogs. Methods: The fecal microbiota was assessed by 454-pyrosequencing. Sequence analysis was performed using QIIME v1.7. Data were analyzed using the bioinformatics software PICRUSt to predict the functional capabilities of bacteria within each sample. Serum metabolites were profiled using a GC-TOF/MS platform and analyzed using MetaboAnalyst 2.0. The disease status was assessed using the clinical indices for dogs with IBD. Results: Fecal bacterial diversity was lower in dogs with IBD. Principle coordinate analysis based on unweighted unifrac distance metric revealed clustering between healthy control dogs and dogs with IBD (ANOSIM, p<0.05), but not between pre- and post-treatment groups. Linear discriminate analysis effects size (LEfSe) detected 25 differentially abundant bacterial clades. While Gammaproteobacteria were overrepresented, Erysipelotrichia, Clostridia, and Bacteroidia were underrepresented in dogs with IBD. No differentially abundant bacterial clades were found between pre- and post-treatment. PICRUSt revealed altered amino acid metabolism and membrane transport system of the microbiota in dogs with IBD. Serum concentrations of 3-Hydroxybutyrate, hexuronic acid, ribose, gluconolactone were significantly increased in dogs with IBD. Only the concentration of gluconolactone was significantly decreased after treatment. Metabolite set enrichment analysis detected over-representation of the pentose phosphate pathway in dogs with IBD. Although clinical improvement was observed after 21 days of immunosuppressive therapy, this was not accompanied by significant changes in the fecal microbiome or in serum metabolite concentrations. Conclusions: In this study, an intestinal dysbiosis and altered serum metabolite concentrations were observed in dogs with IBD. The pattern of dysbiosis in dogs with IBD resembles the dysbiosis observed in humans with IBD. These changes may suggest the presence of oxidative stress and the functional alteration of GI microbiota in dogs with IBD.
Mo1702 Validation of Piroxicam-Accelerated Colitis in the Interleukin-10 Knock out Mouse - A Preclinical Model Mimicking Human Inflammatory Bowel Disease Kristine Holgersen, Peter H. Kvist, Axel K. Hansen, Thomas L. Holm Background: Oral piroxicam administration is a method for induction of enterocolitis in interleukin-10 knock out (IL-10 k.o.) mice. The model combines a dysregulated immune response against the gut microbiota with a decreased mucosal integrity. The piroxicamaccelerated colitis (PAC) IL-10 k.o. model develops histopathological changes in both colon and ileum and the innate immune system seems to be the main driver of disease. However, the characterisation of the underlying pathogenic mechanisms needs further investigation. Additionally, in order to assess the translational value of the PAC IL-10 k.o. model, its ability to respond to existing IBD therapies is evaluated. Methods: C57BL6/J IL-10 k.o. mice received piroxicam in the chow throughout the prophylactic study period i.e. day 0-16. Model qualification was performed by examining efficacy of anti-TNFα monoclonal antibody (mAb), anti-IL-12/23p40 mAb, prednisolone and cyclosporine A. Efficacy was evaluated by body weight loss, disease activity index (DAI) score, colonic weight:length (W:L) ratio and histology. To evaluate the importance of immune cell subsets in disease pathogenesis specific cell types were depleted by treatment with anti-CD4 mAb, anti-CD8 mAb and liposomal encapsulated clodronate. T cell co-stimulation was blocked by CTLA4-Ig and cytokine profiling ELISAs were performed on colon homogenates. Results: Prophylactic treatment with anti-IL-12/23p40 mAb and cyclosporine A prevented disease in PAC IL-10 k.o. mice. The two treatments reduced weight loss, decreased DAI score and reduced W:L ratio and histology score compared to control treatments. Additionally, anti-IL-12/23p40 mAb and cyclosporine A blocked the production of IFNγ, IL-17 and MPO in colon. Anti-TNFα treatment ameliorated clinical disease of PAC IL-10 k.o. mice and a tendency towards reduced colonic pathology was observed. No effect of prednisolone administration was detected. Depletion of CD8+ T cells significantly increased mortality during early disease, whereas treatment with anti-CD4+ and CTLA4-Ig had no effect on disease development in the PAC IL-10 k.o. model. Clodronate treatment caused a 97-98 % decrease in the frequency of splenic macrophages and induced a significant drop in body weight. Nevertheless, the depletion of macrophages was associated with a significant reduction in colonic inflammation. Conclusion: Reference drugs with known efficacy in IBD were efficacious in the PAC IL-10 k.o. model. The ameliorative drugs reduced the production of MPO, IL-17 and IFNγ in the colon, which indicates a role of these cytokines in disease pathogenesis. CD8+ T cells seem to protect against disease in the PAC IL-10 k.o. model. In contrast, our data indicate that macrophages are a main driver of the enterocolitis, whereas CD4+ T cells are not.
Mo1700 Novel Murine Heterotopic Transplant Model of Intestinal Fibrosis Christian Lutz, Gabriela Bollmann, Michaela Caj, Michael Fried, Gerhard Rogler, Martin Hausmann BACKGROUND: Severe mucosal tissue damage requiring efficient wound healing is a main feature of inflammatory bowel disease (IBD) but excessive tissue repair promotes fibrosis. The clinical investigation of fibrosis is confined to the limited amount of biological material available from patients. This makes the establishment of a new animal model a highly desirable goal. We investigated whether intestinal fibrosis occurs after heterotopic transplantation of small bowel resections in mice. METHODS: Donor (B6) small bowel resections were transplanted subcutaneously into the neck of recipients (B6). Grafts were explanted 1, 2, 3, 4, 5, 6, 7, 14 and 21 days after transplantation. Collagen layer thickness was determined from 16 places in representative areas and calculated by Kruskal-Wallis one way analysis of variance on ranks. RESULTS: Heterotopic intestinal transplants remained viable for 21 days. Rapid loss of crypt structures at day one after intestinal transplantation was followed by lymphocyte infiltration and obliteration of the intestinal lumen by fibrous tissue at day 21. Histologic cross sections were stained with EvG to highlight collagen. Freshly isolated samples exhibited very little collagen staining at the submucosal border. In contrast, collagen layer thickness in allografts was increased at day seven after transplantation. Increased collagen
Mo1703 Schistosoma Mansoni Helminth Egg Antigens (SEA) and Their Protective Effect on Colitis in a Murine Transfer Model Marthe Heylen, Nathalie E. Ruyssers, Sara Nullens, Joris G. De Man, Dorien M. Schrijvers, Ilse Van Brussel, Gabriele Schramm, Paul A. Pelckmans, Tom G. Moreels, Benedicte Y. De Winter Introduction: Helminth-based therapy as a novel treatment strategy for inflammatory bowel disease is a well-established concept by now, however the search for immunomodulatory helminth-derived compounds and their mechanisms of action is still going on. Our aim was to determine whether a treatment with Schistosoma mansoni Egg Antigens (SEA) could influence the development and course of colitis using a murine colitis transfer model.
properties of IBD98E. Methods. Acute DSS-induced colitis was performed by adding 3% DSS in drinking water in WT mice for 5 days. After this time, DSS was replaced with regular water and treated intrarectally with or without IBD98E for 6 days. Body weight, disease activity index were monitored daily, while endoscopic analysis was performed at day 0, 5, 9 and 11. At the sacrifice, a histological analysis was evaluated. Caco-2 cell line was used to test in vitro the effects of IBD98E. The cell viability and proliferation after treatment with of IBD98E were evaluated respectively by XTT test and BrdU incorporation. Wound scratch was made in the monolayers of cells incubated for 24h with epidermal growth factor (EGF) 100 ng/ml or IBD98E with or without mitomycin C treatment to inhibit cell proliferation. Photographs of the wounded area were taken at time 0 and after 24 h of the stimulation, and the percentage wound closure was calculated. Results. Colitic mice treated with IBD98E showed faster clinical recovery, significant reduced endoscopic signs of mucosal inflammation (p<0,01) with mild bleeding after 4 and 6 days of the treatment compared to those receiving saline. Furthermore, the histological analysis revealed a minor ulceration score in mice treated with IBD98E. In vitro, IBD98E treatment enhanced cell viability promoting proliferation. Indeed, a significant increased of BrdU incorporation (p<0,05) as well as a wound closure (p<0,05) was found after 24 h in the cells stimulated with IBD98E compared to untreated cells, while no significant differences were observed between IBD98E and EGF. Interestingly, no wound closure was found in response to IBD98E after pre-treatment with mitomycin C, indicating that IBD98E promotes proliferation but does not affect the migration of epithelial cells. Conclusions. Overall these data demonstrate that sodium hyaluronate gel application is safe and favors the clinical and endoscopic outcomes of colitis reducing mucosal bleeding and promoting mucosal wound healing through proliferation of epithelial cells. Therefore IBD98E could represent a new potential treatment to promote mucosal repair in UC patients.