26 5th International Meeting on Inflammatory Bowel Diseases, Capri, April 8 10, 2010 in primary human dendritic cells (DCs). The presence of CD- related mutations in NOD2 or ATG16L1 results in abrogation of autophagy induction, defective bacterial handling and antigen presentation. Persistence or aberrant immune responses to commensal bacteria could trigger inflammatory responses in CD. The mechanism by which NOD2, or other pathogen recognition receptors, induces autophagy remains unknown. In this work I am defining how NOD2 triggers autophagy in DCs using a combination of proteomic approaches. Aim: To define the molecular mechanism by which NOD2 induces autophagy in human cells Materials and Methods: 293 cells stably expressing EGFP-NOD2 and EGFP-1007fsinsC NOD2 have been created and validated as functional in terms of IL8 production, MAPK phosphorylation and LC3 redistribution on NOD2 stimulation. These have been used to conduct a proteomic analysis of NOD2 interacting proteins by pull-down of EGFP-NOD2 and mass spectrometry analysis using LC-MS/MS and Q-TOF. NOD2 signalling has been investigated in primary human DCs using a novel phosphoproteomic technique capable of detecting low abundance signalling proteins in primary cells. The function of identified proteins in NOD2 mediated autophagy induction is being established using siRNAs to knockdown individual targets prior to NOD2 triggering and assessment of autophagy. Results: Proteomic analysis has revealed NOD2 interacting proteins and signalling mediators whose function in NOD2 autophagy induction in primary human cells is being validated using siRNA knockdown and functional experiments. Conclusions: We have established that NOD2 induces au- tophagy. This is required for bacterial handling and antigen presentation in human DCs and is lost in the presence of CD- related NOD2 and ATG16L1 risk variants. This work investigates the molecular mechanism by which NOD2 induces autophagy in DCs using a combination of proteomic approaches delineating both NOD2 interacting proteins and signalling cascade in induction of autophagy. S19 Granulocyte-macrophage colony-stimulating factor elicits bone marrow-derived cells that promote efficient colonic mucosal healing D. Velin 2 , E. Bernasconi 2 , L. Favre 2 , M.H. Maillard 2 , H. Bouzourene 4 , J. Parkinson 1 , P. Michetti 3 . 1 Berlex Biosciences, Richmond, CA, USA, 2 Division of Gastroenterology and Hepatology, CHUV, Lausanne University, Lausanne, Switzerland, 3 Gastro-ent´ erologie La Source-Beaulieu, Lausanne, Switzerland, 4 Institute of Pathology, CHUV, Lausanne University, Lausanne, Switzerland Background and Aims: Granulocyte-macrophage colony- stimulating factor (GM-CSF) therapy is effective in treating some Crohn’s disease (CD) patients and protects mice from colitis induced by dextran sulfate sodium (DSS) administration. However, its mechanisms of action remain elusive. We hypothesized that GM-CSF affects intestinal mucosal repair. Methods: DSS colitic mice were treated with daily pegylated GM-CSF or saline, and clinical, histological and inflammatory parameters were kinetically evaluated. Further, the role of bone marrow-derived cells in the impact of GM-CSF therapy on DSS colitis was addressed using cell transfers. Results: GM-CSF therapy reduced clinical signs of colitis and the release of inflammatory mediators. GM-CSF therapy improved mucosal repair, with faster ulcer reepithelialization, accelerated hyperproliferative response of epithelial cells in ulcer-adjacent crypts and lower colonoscopic ulceration scores in GM-CSF-administered mice relative to untreated mice. We observed that GM-CSF-induced promotion of mucosal repair is timely associated with a reduction in neutrophil numbers and increased accumulation of CD11b + monocytic cells in colon tissues. Importantly, transfer of splenic GM-CSF- induced CD11b + myeloid cells into DSS-exposed mice improved colitis, and lethally irradiated GM-CSF receptor-deficient mice reconstituted with wild-type bone marrow cells were protected from DSS-induced colitis upon GM-CSF therapy. Lastly, GM-CSF- induced CD11b + myeloid cells were shown to promote in vitro wound repair. Conclusions: Our study shows that GM-CSF-dependent stimula- tion of bone marrow-derived cells during DSS-induced colitis accelerates colonic tissue repair. These data provide one putative mechanism for the observed beneficial effects of GM- CSF therapy in human Crohn’s disease. S20 The lymphatic system undergoes lymphangiogenesis and governs an inflammation-dependent mechanism for leukocyte transmigration in IBD patients C. Correale 2 , S. Vetrano 2 , F. Tognoli 1 , A. Spinelli 2 , A. Malesci 2 , E. Dejana 1 , S. Danese 2 . 1 IFOM, Milan, Italy, 2 Istituto Clinico Humanitas, Milan, Italy Background: The exit of antigen presenting cells and lymphocytes from the inflamed intestinal mucosa and their entry into the mesenteric lymph nodes is essential for both the initiation and maintenance of chronic intestinal inflammation. How immune cells transmigrate the lymphatic endothelium in the gut is unknown, and little information is available on the activation state of the lymphatic microvasculature in inflammatory bowel disease (IBD). Aims: We investigated the activation state of the lymphatic system in Crohn’s disease (CD) and ulcerative colitis (UC) by quantifying mucosal lymphatic vascularization, by investi- gating cell adhesion molecule expression, and isolating and characterizing in vitro the functional properties of primary human intestinal lymphatic endothelial cells (HILEC). Control (n = 8), CD (n = 10) and UC (n = 10) colonic mucosa were stained for the lymphatic antigen D2-40, VCAM-1 and ICAM-1, and vessels quantified by morphometry. HILEC cultures were characterized in vitro by fluorescence with anti-Prox-1 and anti-Pal-E antibodies. HILEC were stimulated with TNF-a and investigated for VCAM-1 and ICAM-1 expression by flow cytometry. Peripheral blood T-cell (PBT) and dendritic cell (DC) adhesion and migration were measured by adhesion and migration assays. Materials and Methods: Microvessel lymphatic density was significantly (p < 0.05) higher in CD (22±2) and UC (16±2) compared to control mucosa (7±1). Purity of HILEC was determined by 99% expression of Prox-1 and absence of Pal-E. In vivo, lymphatic vessels displayed enhanced expression of VCAM-1 and ICAM-1 in both CD and UC compared to controls. Resting HILEC expressed undetectable levels of VCAM-1 (0.1%) and physiological levels (42%) of ICAM-1. After TNF-a stimulation both VCAM-1 (58%) and ICAM-1 (92%) were up-regulated, and significantly (p < 0.01) bound more PBT (6 fold increase) and DC (4 fold increase) than resting HIMEC. Adhesion for PBT and DC was VCAM-1 (p < 0.05) but not ICAM-1 dependent, while migration was both VCAM-1 (p < 0.01) and ICAM-1 (p < 0.05) dependent for both cell types, as demonstrated by the use of blocking antibodies against each CAM. Finally, TNF-a stimulated HILEC demonstrate increased production of key chemokines for leukocyte transmigration including IL-8, RANTES, MCP-1, IP-10.