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P265 - Induction and modulation of T cell regulatory function by intestinal fibroblasts: inhibitory effect of intestinal inflammation
Abstracts of the 4th Congress of ECCO
the European Crohn’s and Colitis Organisation
Results: Among the patients enrolled in the study, 8 patients were heterozygous for a variant TPMT allele (2.6%), while the rest had a wild-type gene. Frequencies of deficient alleles TPMT*2, TPMT*3A, and TPMT*3C were 0.6%, 0.6% and 2.6%. No TPMT *3B allele was found. Conclusion: TPMT*3C was the most frequently ocurring nonfunctional TPMT allele in Croatian IBD population. Although predominant allele is obvious in this set of patients, regarding to relatively lower frequency of TPMT polymorphisms we need further investigation in cohort of healthy controls prior definite conclusion about benefit of pharmacogenetically guided dosing in our population. P264 Extra-intestinal manifestations in IBD are associated with specific mucosal inflammatory genes expression profiles M.M. Diculescu1 *, R. Iacob1 , D. Iancu2 , L. Barbarii2 , B. Cotruta3 , S. Iacob3 , S. Gologan1 , C. Baceanu1 , A. Catuneanu1 , C. Gheorghe3 , L. Gheorghe1 , S. Iobagiu4 . 1 University of Medicine and Pharmacy “Carol Davila”, Bucharest, Romania, 2 National Institute of Legal Medicine “Mina Minovici”, Bucharest, Romania, 3 Fundeni Clinical Institute, Bucharest, Romania, 4 University of Medicine and Pharmacy “Iuliu Hatieganu”, Cluj-Napoca, Romania Background and Aim: The presence of extra-intestinal manifestations is an important feature associated with different IBD phenotypes. The aim of this study was to investigate whether a specific mucosal inflammatory genes expression profile can be identified in patients with IBD having also extra-intestinal manifestations. Methods: Sixty-eight IBD patients have been included in the analysis (37 CD and 31 UC). For each patient biopsies were taken during colonoscopy from inflamed tissue (L) and healthy colonic mucosa (N). RNA isolated from the tissue samples was processed using a multiplex ligation-dependent probe amplification (MLPA) protocol and an Inflammation mRNA dedicated kit (SALSA RT-MLPA R009, MRC Holland). The screening protocol comprises probes for a set of 40 mRNA molecules: cytokines, receptors, signal transduction molecules, transcription factors. PCR products were detected by capillary electrophoresis on a 3100 Avant Genetic Analyzer (Applied Biosystems). The PARN gene was used as reference to normalise the signals. Normalized signals were compared for each gene between CD and UC patients for L and N biopsies respectively, using Mann Whitney U test. Significant variables were included in a logistic regression analysis in order to identify independent variables associated with the presence of extraintestinal manifestations in patients with IBD. Results: Twenty-one patients (30.6%) had extra-intestinal manifestations including: arthritis, spondylitis/sacroiliitis, primary sclerosing cholangitis, uveitis, pioderma gangrenosum, erythema nodosum etc. In L biopsies from patients having extra-intestinal manifestations significantly different expression levels were found for IL1RN (p = 0.02) and SCYA8 (p = 0.02). In N biopsies from patients with with extra-intestinal manifestations significantly different expression levels were found for THBS1 (p = 0.03) and BMI1 (0.002). The multivariate logistic regression analysis has indicatd that lower expression levels of THBS1 and BMI1 in non-inflammed colonic mucosa are independent factors associated with the presence of extraintestinal manifestations in patients with IBD. Based on the logistic regression equation, a prediction model has been constructed using the two independent variables. The model has a c-statistic parameter of 0.77 for predicting the presence of extraintestinal manifestations, and a cutt-off value of 0.28 might have a clinical use. Conclusion: MLPA method is a sensitive and effective screening tool able to characterise the inflammatory genes expression profiles in patients with IBD. A specific mucosal inflammatory
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genes expression profile can be identified in patients with IBD having also extra-intestinal manifestations. This profile is characterized by lower expression levels of THBS1 and BMI1 in non-inflammed colonic mucosa. P265 Induction and modulation of T cell regulatory function by intestinal fibroblasts: inhibitory effect of intestinal inflammation F. Scaldaferri IV1 *, F. Rieder2 , A. Schirbel2 , G. West2 , S. Rutella1 , S. Danese3 , C. Fiocchi2 . 1 Catholic University of Rome, Rome, Italy, 2 the Cleveland Clinic Foundation Lerner Research Institute, Cleveland, OH, USA, 3 Humanitas University of Milan, Rozzano, Milan, Italy Background: Intestinal fibroblasts are active players in intestinal immune homeostasis, but their interaction with CD4+CD25+FoxP3+ T cells (Tregs) has not been explored. We investigated whether human intestinal fibroblasts (HIF) modulate Treg number and/or function, and how this interaction may be altered in the presence of conditions mimicking inflammation. Results: CD4+CD25+T cells purified from peripheral blood (PB) T cells were cultured alone or in the presence of HIF, and Treg numbers assessed after 4 days. The simple contact with HIF increased the viability of Tregs compared to cells cultured alone (37.1 vs 9.1%, respectively). This protective anti-apoptotic effect was enhanced if IL-2 was present (49.4%), but reduced (14.3%) when a transwell system prevented physical contact between Tregs and HIF. Furthermore, co-culture with HIF maintained FoxP3 mRNA expression level, which essentially disappeared if T cells were cultured alone. In addition to extending the lifespan of PB Tregs, HIF induced Tregs de novo from naive CD4+CD45RO T cells through physical interaction; Treg induction increased if IL-2 and proinflammatory cytokines were added (2.7 and 4.1%, respectively) to the co-culture, and was accompanied by a dramatic upregulation of HIF PDL-1 expression (from 5 to 85%). In contrast to PB-derived Tregs, which do not proliferate, HIF-induced Tregs proliferated strongly in response to TCR activation but still exhibited a potent suppressive activity, as shown by strong inhibition of autologous CD4+CD25 T cell proliferation. Interestingly, the suppressive activity of HIFinduced Tregs was substantially reduced (from 60% to 30%) if induction occurred in the presence of TNF-a and IL-1b. TCR-activated HIF-induced Tregs produced low amounts of IFN-g, TNF-a, IL2, IL5 and IL10 compared to autologous CD4+CD25 cells. However, if TNF-a and IL-1b were present during the induction period, the resulting Tregs produced much higher levels of cytokines than those generated by activated autologous CD4CD25 cells, suggesting a switch to effector T cells. Conclusion: Intestinal fibroblasts contribute to Tregs homeostasis by extending their lifespan and inducing the de novo generation of functional Tregs from naive T cells, apparently through a PDL1-dependent mechanism. Pro-inflammatory cytokines, while enhancing generation of HIF-induced Tregs, apparently promote a switch from a suppressor to an effector T cell phenotype. These results suggest that mesenchymal cells promote mucosal homeostasis by supporting and expanding local Tregs, but this beneficial effect is lost in the presence of inflammation, which may help explain the inadequate Treg activity found in IBD.
the European Crohn’s and Colitis Organisation
Results: Among the patients enrolled in the study, 8 patients were heterozygous for a variant TPMT allele (2.6%), while the rest had a wild-type gene. Frequencies of deficient alleles TPMT*2, TPMT*3A, and TPMT*3C were 0.6%, 0.6% and 2.6%. No TPMT *3B allele was found. Conclusion: TPMT*3C was the most frequently ocurring nonfunctional TPMT allele in Croatian IBD population. Although predominant allele is obvious in this set of patients, regarding to relatively lower frequency of TPMT polymorphisms we need further investigation in cohort of healthy controls prior definite conclusion about benefit of pharmacogenetically guided dosing in our population. P264 Extra-intestinal manifestations in IBD are associated with specific mucosal inflammatory genes expression profiles M.M. Diculescu1 *, R. Iacob1 , D. Iancu2 , L. Barbarii2 , B. Cotruta3 , S. Iacob3 , S. Gologan1 , C. Baceanu1 , A. Catuneanu1 , C. Gheorghe3 , L. Gheorghe1 , S. Iobagiu4 . 1 University of Medicine and Pharmacy “Carol Davila”, Bucharest, Romania, 2 National Institute of Legal Medicine “Mina Minovici”, Bucharest, Romania, 3 Fundeni Clinical Institute, Bucharest, Romania, 4 University of Medicine and Pharmacy “Iuliu Hatieganu”, Cluj-Napoca, Romania Background and Aim: The presence of extra-intestinal manifestations is an important feature associated with different IBD phenotypes. The aim of this study was to investigate whether a specific mucosal inflammatory genes expression profile can be identified in patients with IBD having also extra-intestinal manifestations. Methods: Sixty-eight IBD patients have been included in the analysis (37 CD and 31 UC). For each patient biopsies were taken during colonoscopy from inflamed tissue (L) and healthy colonic mucosa (N). RNA isolated from the tissue samples was processed using a multiplex ligation-dependent probe amplification (MLPA) protocol and an Inflammation mRNA dedicated kit (SALSA RT-MLPA R009, MRC Holland). The screening protocol comprises probes for a set of 40 mRNA molecules: cytokines, receptors, signal transduction molecules, transcription factors. PCR products were detected by capillary electrophoresis on a 3100 Avant Genetic Analyzer (Applied Biosystems). The PARN gene was used as reference to normalise the signals. Normalized signals were compared for each gene between CD and UC patients for L and N biopsies respectively, using Mann Whitney U test. Significant variables were included in a logistic regression analysis in order to identify independent variables associated with the presence of extraintestinal manifestations in patients with IBD. Results: Twenty-one patients (30.6%) had extra-intestinal manifestations including: arthritis, spondylitis/sacroiliitis, primary sclerosing cholangitis, uveitis, pioderma gangrenosum, erythema nodosum etc. In L biopsies from patients having extra-intestinal manifestations significantly different expression levels were found for IL1RN (p = 0.02) and SCYA8 (p = 0.02). In N biopsies from patients with with extra-intestinal manifestations significantly different expression levels were found for THBS1 (p = 0.03) and BMI1 (0.002). The multivariate logistic regression analysis has indicatd that lower expression levels of THBS1 and BMI1 in non-inflammed colonic mucosa are independent factors associated with the presence of extraintestinal manifestations in patients with IBD. Based on the logistic regression equation, a prediction model has been constructed using the two independent variables. The model has a c-statistic parameter of 0.77 for predicting the presence of extraintestinal manifestations, and a cutt-off value of 0.28 might have a clinical use. Conclusion: MLPA method is a sensitive and effective screening tool able to characterise the inflammatory genes expression profiles in patients with IBD. A specific mucosal inflammatory
S115
genes expression profile can be identified in patients with IBD having also extra-intestinal manifestations. This profile is characterized by lower expression levels of THBS1 and BMI1 in non-inflammed colonic mucosa. P265 Induction and modulation of T cell regulatory function by intestinal fibroblasts: inhibitory effect of intestinal inflammation F. Scaldaferri IV1 *, F. Rieder2 , A. Schirbel2 , G. West2 , S. Rutella1 , S. Danese3 , C. Fiocchi2 . 1 Catholic University of Rome, Rome, Italy, 2 the Cleveland Clinic Foundation Lerner Research Institute, Cleveland, OH, USA, 3 Humanitas University of Milan, Rozzano, Milan, Italy Background: Intestinal fibroblasts are active players in intestinal immune homeostasis, but their interaction with CD4+CD25+FoxP3+ T cells (Tregs) has not been explored. We investigated whether human intestinal fibroblasts (HIF) modulate Treg number and/or function, and how this interaction may be altered in the presence of conditions mimicking inflammation. Results: CD4+CD25+T cells purified from peripheral blood (PB) T cells were cultured alone or in the presence of HIF, and Treg numbers assessed after 4 days. The simple contact with HIF increased the viability of Tregs compared to cells cultured alone (37.1 vs 9.1%, respectively). This protective anti-apoptotic effect was enhanced if IL-2 was present (49.4%), but reduced (14.3%) when a transwell system prevented physical contact between Tregs and HIF. Furthermore, co-culture with HIF maintained FoxP3 mRNA expression level, which essentially disappeared if T cells were cultured alone. In addition to extending the lifespan of PB Tregs, HIF induced Tregs de novo from naive CD4+CD45RO T cells through physical interaction; Treg induction increased if IL-2 and proinflammatory cytokines were added (2.7 and 4.1%, respectively) to the co-culture, and was accompanied by a dramatic upregulation of HIF PDL-1 expression (from 5 to 85%). In contrast to PB-derived Tregs, which do not proliferate, HIF-induced Tregs proliferated strongly in response to TCR activation but still exhibited a potent suppressive activity, as shown by strong inhibition of autologous CD4+CD25 T cell proliferation. Interestingly, the suppressive activity of HIFinduced Tregs was substantially reduced (from 60% to 30%) if induction occurred in the presence of TNF-a and IL-1b. TCR-activated HIF-induced Tregs produced low amounts of IFN-g, TNF-a, IL2, IL5 and IL10 compared to autologous CD4+CD25 cells. However, if TNF-a and IL-1b were present during the induction period, the resulting Tregs produced much higher levels of cytokines than those generated by activated autologous CD4CD25 cells, suggesting a switch to effector T cells. Conclusion: Intestinal fibroblasts contribute to Tregs homeostasis by extending their lifespan and inducing the de novo generation of functional Tregs from naive T cells, apparently through a PDL1-dependent mechanism. Pro-inflammatory cytokines, while enhancing generation of HIF-induced Tregs, apparently promote a switch from a suppressor to an effector T cell phenotype. These results suggest that mesenchymal cells promote mucosal homeostasis by supporting and expanding local Tregs, but this beneficial effect is lost in the presence of inflammation, which may help explain the inadequate Treg activity found in IBD.