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MODULATION OF T CELL REGULATORY FUNCTION BY INTESTINAL FIBROBLASTS: WHERE INFLAMMATION INHIBITS IMMUNE-REGULATION
Abstracts / Digestive and Liver Disease 41S (2009), S1–S167 OC.04.1 SMAD7 EXPRESSION IN T CELLS PROTECTS FROM COLITIS-ASSOCIATED COLORECTAL CANCER M.C. Fantini ∗ ,1 , A. Rizzo 1 , D. Fina 1 , R. Caruso 1 , C. Stolfi 1 , M. Sarra 1 , C. Becker 2 , T.T. MacDonald 3 , F. Pallone 1 , M. Neurath 2 , G. Monteleone 1 1 University Tor Vegata, Roma; 2 University Johannes Gutenberg, Mainz; 3 Bart’s and The London School of Medicine and Dentistry, London
Background and aim: Prolonged inflammation of the gut has been associated with enhanced colorectal cancer (CC) risk. This for example occurs in patients with long-standing inflammatory bowel disease (IBD). However, studies in other systems have shown that a proper immune response contributes to limit tumor growth and diffusion. Transforming growth factor (TGF)-beta1, a cytokine involved in the negative control of the mucosal immune system, has been shown to prevent CC. Aim: Since in IBD there is high expression of Smad7, an inhibitor of TGF-beta1, we evaluated whether Smad7-mediated block of the TGF-beta1 signaling in T cells could promote CC initiation and growth. Material and methods: Wild type and transgenic mice over-expressing Smad7 under control of the CD2 promoter (Smad7tg) were treated with axozymethane (AOM) and three cycles of oral dextrane sulphate sodium (DSS) to induce colitis-associated CC. Tumor number and size were assessed by endoscopy. Colitis and dysplasia were graded by histology and expression of proinflammatory cytokines was evaluated in mononuclear cells from tumors and tumor free areas by flow-cytometry and real-time pcr. Granzynes B, perforin-1, and FasL mRNA expression was evaluated by real-time pcr. Results: As expected Wt mice developed large and multiple tumors after AOM/DSS treatment. By contrast, only few and small tumors were seen in Smad7tg mice, despite these animals developed a more pronounced colitis. Consistently, higher expression of IFN-gamma, IL-6 and IL-17 was found in the tumor free areas of the Smad7tg mice. Further flow-cytometry analysis of tumor-infiltrating T cells showed a higher expression of high IFN-gamma in both CD4+ and CD8+ T cells from Smad7Tg in comparison to Wt mice. Analysis of CD8+ T-cellrelated cytotoxic molecules showed higher expression of Perforin-1, Granzyme B and FasL in both tumors and tumor-free areas of the Smad7tg in comparison to the Wt animals. Conclusions: T cell-specific Smad7 over-expression results in a more severe colitis that protects against CC. Our data suggest that high expression of IFN-gamma and cytotoxicity sustained by high Smad7 in T cells could play a role in the regulation of anti-tumor immune responses in the gut. # J. GI Oncology 1. Basic science
OC.04.2 MODULATION OF T CELL REGULATORY FUNCTION BY INTESTINAL FIBROBLASTS: WHERE INFLAMMATION INHIBITS IMMUNE-REGULATION F. Scaldaferri ∗ ,1 , F. Rieder 2 , A. Schirbel 2 , G. West 2 , S. Rutella 1 , A. Gasbarrini 1 , S. Danese 3 , C. Fiocchi 2 1 Catholic
University - Gemelli Hospital, Roma; 2 The Cleveland Clinic Foundation, Cleveland, USA; 3 Humanitas - University of Milan, Rozzano, Milano Background and aim: Human intestinal fibroblasts (HIF) are active players in intestinal immune homeostasis, but their interaction with CD4+CD25+FoxP3+ T cells (Tregs) has not been explored. We investigated whether HIF modulate Treg number and/or function, and how this interaction may be altered in the presence of conditions mimicking inflammation.
S19
Material and methods: CD4+CD25+T cells and CD4+CD45ROnaïve T cells (naïve T cells) were purified from peripheral blood (PB) T cells. HIF are primary cells isolated from human intestinal surgical specimens. Results: CD4+CD25+T cells were cultured alone or in the presence of HIF, and Treg numbers assessed after 4 days. The presence of HIF increased the viability of Tregs compared to cells cultured alone (37.1 vs 9.1%), or without direct contact (transwell system, 14.3%). In addition to that, HIF induced Tregs de novo from naive T cells; Treg induction increased in presence of IL-2 and pro-inflammatory cytokines (2.7 and 4.1%, respectively) and was accompanied by a dramatic upregulation of HIF PDL-1 expression (from 5 to 85%). In contrast to PB-derived Tregs, 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 HIF-induced Tregs was substantially reduced (from 60% to 30%) if induction occurred in the presence of TNF-α and IL-1β. TCR-activated HIF-induced Tregs produced low amounts of IFN-γ, TNF-α, IL2, IL5 and IL10 compared to autologous CD4+CD25- cells. However, if TNF-α and IL-1β were present during the induction period, the resulting Tregs produced much higher levels of cytokine, suggesting a switch to effector T cells. Conclusions: Intestinal fibroblasts contribute to Tregs homeostasis by extending their lifespan and inducing the de novo generation of functional Tregs from naïve 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. # L. Inflammatory bowel diseases 1. Basic science
OC.04.3 FIBROBLAST ACTIVATION PROTEIN EXPRESSION IN CROHN’S DISEASE STRICTURES L. Rovedatti ∗ ,1 , A. Di Sabatino 1 , P. Biancheri 1 , P. Cazzola 1 , T.T. MacDonald 2 , G.R. Corazza 1 1 Clinica Medica I, Fondazione IRCCS Policlinico S. Matteo, Centro Per Lo Studio e La Cura Delle Malattie Infiammatorie Croniche Intestinali, Università di Pavia, Pavia; 2 Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, London
Background and aim: Fibroblast activation protein (FAP), a 170-kDa cell-surface glycoprotein with gelatinase and dipeptidyl-peptidase activity, is an early marker of fibroblast activation, mostly expressed in wound healing tissue or fibrotic disorders. On this basis, we verified its expression in fibrostenosing Crohn’s disease (CD). Material and methods: Colonic surgical specimens were collected from strictures and non-strictured areas of 15 fibrostenosing CD patients, from normal colon of 15 control subjects and from involved areas of 12 ulcerative colitis (UC) patients. FAP and alpha-smooth muscle cell actin (SMA) expression was evaluated by immunohistochemistry on cryostat sections. Both fibroblasts and lamina propria mononuclear cells (LPMCs) were isolated from tissue specimens and analysed by flow cytometry for FAP expression. FAP was also determined by immunoblotting on tissue homogenates or cell lysates. Results: The immunohistochemical analysis showed a significantly higher FAP expression in CD strictures than in CD non-strictured areas, normal colon and UC tissue. No significant difference was found between CD non-stricured areas, normal colon and UC tissue. In CD strictures, FAP was mostly localised in the submucosa and expressed by SMA+ cells. However, a higher percentage of FAP+ cells was also
Background and aim: Prolonged inflammation of the gut has been associated with enhanced colorectal cancer (CC) risk. This for example occurs in patients with long-standing inflammatory bowel disease (IBD). However, studies in other systems have shown that a proper immune response contributes to limit tumor growth and diffusion. Transforming growth factor (TGF)-beta1, a cytokine involved in the negative control of the mucosal immune system, has been shown to prevent CC. Aim: Since in IBD there is high expression of Smad7, an inhibitor of TGF-beta1, we evaluated whether Smad7-mediated block of the TGF-beta1 signaling in T cells could promote CC initiation and growth. Material and methods: Wild type and transgenic mice over-expressing Smad7 under control of the CD2 promoter (Smad7tg) were treated with axozymethane (AOM) and three cycles of oral dextrane sulphate sodium (DSS) to induce colitis-associated CC. Tumor number and size were assessed by endoscopy. Colitis and dysplasia were graded by histology and expression of proinflammatory cytokines was evaluated in mononuclear cells from tumors and tumor free areas by flow-cytometry and real-time pcr. Granzynes B, perforin-1, and FasL mRNA expression was evaluated by real-time pcr. Results: As expected Wt mice developed large and multiple tumors after AOM/DSS treatment. By contrast, only few and small tumors were seen in Smad7tg mice, despite these animals developed a more pronounced colitis. Consistently, higher expression of IFN-gamma, IL-6 and IL-17 was found in the tumor free areas of the Smad7tg mice. Further flow-cytometry analysis of tumor-infiltrating T cells showed a higher expression of high IFN-gamma in both CD4+ and CD8+ T cells from Smad7Tg in comparison to Wt mice. Analysis of CD8+ T-cellrelated cytotoxic molecules showed higher expression of Perforin-1, Granzyme B and FasL in both tumors and tumor-free areas of the Smad7tg in comparison to the Wt animals. Conclusions: T cell-specific Smad7 over-expression results in a more severe colitis that protects against CC. Our data suggest that high expression of IFN-gamma and cytotoxicity sustained by high Smad7 in T cells could play a role in the regulation of anti-tumor immune responses in the gut. # J. GI Oncology 1. Basic science
OC.04.2 MODULATION OF T CELL REGULATORY FUNCTION BY INTESTINAL FIBROBLASTS: WHERE INFLAMMATION INHIBITS IMMUNE-REGULATION F. Scaldaferri ∗ ,1 , F. Rieder 2 , A. Schirbel 2 , G. West 2 , S. Rutella 1 , A. Gasbarrini 1 , S. Danese 3 , C. Fiocchi 2 1 Catholic
University - Gemelli Hospital, Roma; 2 The Cleveland Clinic Foundation, Cleveland, USA; 3 Humanitas - University of Milan, Rozzano, Milano Background and aim: Human intestinal fibroblasts (HIF) are active players in intestinal immune homeostasis, but their interaction with CD4+CD25+FoxP3+ T cells (Tregs) has not been explored. We investigated whether HIF modulate Treg number and/or function, and how this interaction may be altered in the presence of conditions mimicking inflammation.
S19
Material and methods: CD4+CD25+T cells and CD4+CD45ROnaïve T cells (naïve T cells) were purified from peripheral blood (PB) T cells. HIF are primary cells isolated from human intestinal surgical specimens. Results: CD4+CD25+T cells were cultured alone or in the presence of HIF, and Treg numbers assessed after 4 days. The presence of HIF increased the viability of Tregs compared to cells cultured alone (37.1 vs 9.1%), or without direct contact (transwell system, 14.3%). In addition to that, HIF induced Tregs de novo from naive T cells; Treg induction increased in presence of IL-2 and pro-inflammatory cytokines (2.7 and 4.1%, respectively) and was accompanied by a dramatic upregulation of HIF PDL-1 expression (from 5 to 85%). In contrast to PB-derived Tregs, 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 HIF-induced Tregs was substantially reduced (from 60% to 30%) if induction occurred in the presence of TNF-α and IL-1β. TCR-activated HIF-induced Tregs produced low amounts of IFN-γ, TNF-α, IL2, IL5 and IL10 compared to autologous CD4+CD25- cells. However, if TNF-α and IL-1β were present during the induction period, the resulting Tregs produced much higher levels of cytokine, suggesting a switch to effector T cells. Conclusions: Intestinal fibroblasts contribute to Tregs homeostasis by extending their lifespan and inducing the de novo generation of functional Tregs from naïve 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. # L. Inflammatory bowel diseases 1. Basic science
OC.04.3 FIBROBLAST ACTIVATION PROTEIN EXPRESSION IN CROHN’S DISEASE STRICTURES L. Rovedatti ∗ ,1 , A. Di Sabatino 1 , P. Biancheri 1 , P. Cazzola 1 , T.T. MacDonald 2 , G.R. Corazza 1 1 Clinica Medica I, Fondazione IRCCS Policlinico S. Matteo, Centro Per Lo Studio e La Cura Delle Malattie Infiammatorie Croniche Intestinali, Università di Pavia, Pavia; 2 Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, London
Background and aim: Fibroblast activation protein (FAP), a 170-kDa cell-surface glycoprotein with gelatinase and dipeptidyl-peptidase activity, is an early marker of fibroblast activation, mostly expressed in wound healing tissue or fibrotic disorders. On this basis, we verified its expression in fibrostenosing Crohn’s disease (CD). Material and methods: Colonic surgical specimens were collected from strictures and non-strictured areas of 15 fibrostenosing CD patients, from normal colon of 15 control subjects and from involved areas of 12 ulcerative colitis (UC) patients. FAP and alpha-smooth muscle cell actin (SMA) expression was evaluated by immunohistochemistry on cryostat sections. Both fibroblasts and lamina propria mononuclear cells (LPMCs) were isolated from tissue specimens and analysed by flow cytometry for FAP expression. FAP was also determined by immunoblotting on tissue homogenates or cell lysates. Results: The immunohistochemical analysis showed a significantly higher FAP expression in CD strictures than in CD non-strictured areas, normal colon and UC tissue. No significant difference was found between CD non-stricured areas, normal colon and UC tissue. In CD strictures, FAP was mostly localised in the submucosa and expressed by SMA+ cells. However, a higher percentage of FAP+ cells was also