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P025 Vedolizumab does not affect the suppressive activity of gut-homing T regulatory cells
S20 patients were more often absent due to hospital or emergency department visits and abdominal pain, whereas more UC patients were absent due to incontinence or fear of it, fear of toilet frequency interrupting work, worry about gas, discomfort or volume of bleeding. Conclusions: IBD significantly impacts the patients’ quality of life. The symptoms, current therapies, and personal experiences of UC and CD patients, however, are different. The differences should be looked into more deeply to be able to offer more individually targeted therapies to UC and CD patients. P025 Vedolizumab does not affect the suppressive activity of gut-homing T regulatory cells E.R. Fedyk1 *, L.-L. Yang1 , T. Wyant1 , V.J. Kadambi1 . 1 Millennium Pharmaceuticals, Inc., Cambridge, United States Background: The alpha4beta7 integrin is a phenotypic marker of memory T lymphocytes that preferentially migrate through the gut. Phenotypically discreet subsets of human gut-homing memory T cells have been identified, including proinflammatory TH17 and anti-inflammatory T regulatory (Treg) cells. The activity of these subsets may influence human gastrointestinal homeostasis and disease. Vedolizumab is an investigational humanised monoclonal antibody that binds to the alpha4beta7 integrin and is being developed for ulcerative colitis and Crohn’s disease. We therefore investigated to determine if vedolizumab modulated the activity of human Treg cells. Methods: Total (CD4+ CD25+ CD127low FoxP3+ ) and gut-homing (alpha4beta7high CD4+ CD25+ CD127low FoxP3+ ) populations of Treg cells were identified by flow cytometry in whole-blood samples from healthy human volunteers (n = 8). Both populations were subsequently isolated to >95% purity from whole blood by immunomagnetic separation and fluorescence-activated cell sorting. Purified human Treg cells were activated by stimulation with anti-CD2, -CD3 and -CD28 beads, and functional activity was assessed by coculturing with responder T cells, activated by prior stimulation with phytohemagglutinin or anti-CD3/CD28 beads. Proliferation of responder cells was measured by flow cytometry. Potential effects of vedolizumab were assessed by coculturing Treg and responder cells with saturating concentrations of vedolizumab. Results: We identified a subset of gut-homing Treg cells that comprise ~2 15% of the total Treg cell population found in peripheral blood. We isolated these cells and demonstrated that they exhibit suppressive activity that is typical of Treg cells in vitro. Specifically, a coculture of activated responder T cells with the gut-homing Treg subset inhibited proliferation of responder T cells (ie, suppression of proinflammatory activity by Treg). This suppressive activity was not affected by exposure to vedolizumab; incubation of the gut-homing Treg subset with saturating exposures of vedolizumab did not affect the suppression of responder T cells compared with vehicle control or an isotype control antibody. Conclusions: There was no effect of vedolizumab on the suppressive activity of the gut-homing subset of this population found in peripheral blood of healthy volunteers. These data indicate that vedolizumab does not affect potential antiinflammatory activity of human Treg cells residing in the submucosa. P026 Vitamin D enhances macrophage function and improves killing of Crohn’s associated E. coli P. Flanagan1 *, B.J. Campbell1 , J.M. Rhodes1 . 1 University of Liverpool, Gastroenterology, Liverpool, United Kingdom Background: E. coli are increased in Crohn’s disease, have an adherent invasive (AIEC) phenotype and several lines of
Poster presentations evidence suggest a primary pathogenic role. AIEC replicate within macrophages, the function of which is known to be impaired in Crohn’s disease patients [1]. Polymorphisms in the Vitamin D receptor are associated with Crohn’s disease and treatment with Vitamin D reduces the risk of clinical relapse [2]. We postulate Vitamin D supplementation may be efficacious in Crohn’s disease by enhancing macrophage function and improving clearance of AIEC. Methods: We aimed to assess the effect of Vitamin D supplementation on the generation of respiratory burst and the killing of E. coli by macrophages. J774A.1 murine macrophages were infected with representative Crohn’s E. coli isolates, HM605 (colonic) or LF82 (ileal), and the effect of Vitamin D on bacterial survival was assessed using the gentamicin protection assay. Intracellular respiratory burst, determined by generation of fluorescent Dihydrorhodamine from its inactive form, was measured by flow cytometry. Total respiratory burst was quantified by lucigenin mediated chemiluminescence. Results: Compared to untreated control, intra-macrophage E. coli survival was reduced in a dose dependent manner by Vitamin D at concentrations within the normal range for serum levels (9.01%±1.25% at 80nM, p < 0.001, ANOVA, N = 3). At equimolar concentrations, the 1,25-dihydroxylated form had significantly more potent effects on bacterial survival than non-hydroxylated Vitamin D3 (4.05±0.94 fold lower survival at 80nM, p < 0.01, N = 3). Respiratory burst was significantly higher with Vitamin D supplementation compared to untreated control (44.7%±3.9% vs 32.5%±3.8%, p < 0.05, ANOVA, N = 5). Conclusions: Vitamin D supplementation significantly enhances macrophage killing of Crohn’s associated E. coli. Dihydroxylated vitamin D3 is more biologically active than the nonhydroxylated form. A significant increase in respiratory burst is observed and may explain the enhanced killing of AIEC. These findings further support the use of Vitamin D supplementation in the treatment of Crohn’s disease. Reference(s) [1] Flanagan P, et al., (2011), Bacteria in the pathogenesis of inflammatory bowel disease, Biochem Soc Trans, 39;1067 72 [2] Jorgensen S.P., et al., (2010), Clinical trial: vitamin D3 treatment in Crohn’s disease a randomized doubleblind placebo-controlled study. Aliment Pharmacol Ther, 32:377 383 P027 Vitamin D potentiates the immunosuppressive effect of anti-TNF induced regulatory macrophages A. Levin1 *, M. Wildenberg1 , C. Vos2 , J. Brandse3 , J. De Bruyn3 , G. D’Haens3 , G. van den Brink3 . 1 AMC, Tytgat institute, Amsterdam, Netherlands, 2 LUMC, Gastroenterology, Leiden, Netherlands, 3 AMC, Gastroenterology, Amsterdam, Netherlands Background: We have previously shown that anti-TNF-alfa antibodies induce a distinct population of macrophages with immune regulatory and wound healing properties. An environmental factor that has been shown to induce tolerogenicity in dendritic cells is Vitamin D. Interestingly, epidemiological studies have shown that Crohn’s disease patients have a high prevalence of vitamin D deficiency. These results incited us to investigate the role of Vitamin D in antiTNF induced macrophages. The aim of this study was to see if the Vitamin D receptor pathway is involved in the induction of anti-TNF induced macrophages and if Vitamin D can potentiate immunosuppressive effect of these macrophages. Methods: PBMC’s were isolated from peripheral blood of healthy donors. Mixed lymphocyte reactions were established by co-culturing PBMC’s of two healthy individual donors in a 1:1 ratio. Cultures were treated with anti-TNF to induce antiTNF induced macrophages. Inflammatory M1 type macrophages
Poster presentations evidence suggest a primary pathogenic role. AIEC replicate within macrophages, the function of which is known to be impaired in Crohn’s disease patients [1]. Polymorphisms in the Vitamin D receptor are associated with Crohn’s disease and treatment with Vitamin D reduces the risk of clinical relapse [2]. We postulate Vitamin D supplementation may be efficacious in Crohn’s disease by enhancing macrophage function and improving clearance of AIEC. Methods: We aimed to assess the effect of Vitamin D supplementation on the generation of respiratory burst and the killing of E. coli by macrophages. J774A.1 murine macrophages were infected with representative Crohn’s E. coli isolates, HM605 (colonic) or LF82 (ileal), and the effect of Vitamin D on bacterial survival was assessed using the gentamicin protection assay. Intracellular respiratory burst, determined by generation of fluorescent Dihydrorhodamine from its inactive form, was measured by flow cytometry. Total respiratory burst was quantified by lucigenin mediated chemiluminescence. Results: Compared to untreated control, intra-macrophage E. coli survival was reduced in a dose dependent manner by Vitamin D at concentrations within the normal range for serum levels (9.01%±1.25% at 80nM, p < 0.001, ANOVA, N = 3). At equimolar concentrations, the 1,25-dihydroxylated form had significantly more potent effects on bacterial survival than non-hydroxylated Vitamin D3 (4.05±0.94 fold lower survival at 80nM, p < 0.01, N = 3). Respiratory burst was significantly higher with Vitamin D supplementation compared to untreated control (44.7%±3.9% vs 32.5%±3.8%, p < 0.05, ANOVA, N = 5). Conclusions: Vitamin D supplementation significantly enhances macrophage killing of Crohn’s associated E. coli. Dihydroxylated vitamin D3 is more biologically active than the nonhydroxylated form. A significant increase in respiratory burst is observed and may explain the enhanced killing of AIEC. These findings further support the use of Vitamin D supplementation in the treatment of Crohn’s disease. Reference(s) [1] Flanagan P, et al., (2011), Bacteria in the pathogenesis of inflammatory bowel disease, Biochem Soc Trans, 39;1067 72 [2] Jorgensen S.P., et al., (2010), Clinical trial: vitamin D3 treatment in Crohn’s disease a randomized doubleblind placebo-controlled study. Aliment Pharmacol Ther, 32:377 383 P027 Vitamin D potentiates the immunosuppressive effect of anti-TNF induced regulatory macrophages A. Levin1 *, M. Wildenberg1 , C. Vos2 , J. Brandse3 , J. De Bruyn3 , G. D’Haens3 , G. van den Brink3 . 1 AMC, Tytgat institute, Amsterdam, Netherlands, 2 LUMC, Gastroenterology, Leiden, Netherlands, 3 AMC, Gastroenterology, Amsterdam, Netherlands Background: We have previously shown that anti-TNF-alfa antibodies induce a distinct population of macrophages with immune regulatory and wound healing properties. An environmental factor that has been shown to induce tolerogenicity in dendritic cells is Vitamin D. Interestingly, epidemiological studies have shown that Crohn’s disease patients have a high prevalence of vitamin D deficiency. These results incited us to investigate the role of Vitamin D in antiTNF induced macrophages. The aim of this study was to see if the Vitamin D receptor pathway is involved in the induction of anti-TNF induced macrophages and if Vitamin D can potentiate immunosuppressive effect of these macrophages. Methods: PBMC’s were isolated from peripheral blood of healthy donors. Mixed lymphocyte reactions were established by co-culturing PBMC’s of two healthy individual donors in a 1:1 ratio. Cultures were treated with anti-TNF to induce antiTNF induced macrophages. Inflammatory M1 type macrophages