A. Di Sabatino

CC-10004 but not thalidomide or lenalidomide inhibits lamina propria mononuclear cell TNF-α and MMP-3 production in patients with inflammatory bowel disease

BACKGROUND Thalidomide, one of whose activities is to inhibit Tumour Necrosis Factor (TNF)-α production, has been reported to be an effective treatment for refractory inflammatory bowel disease (IBD). TNF-α driven production of matrix metalloproteinase (MMP)-3 by gut lamina propria mononuclear cells (LPMCs) is a major pathway of tissue injury in IBD; however the effect of thalidomide and newer more potent immunomodulatory derivatives on this pathway has not been studied. AIM To investigate the effect of thalidomide, CC-4047 (pomalidomide), CC-5013 (lenalidomide), and CC-10004 (apremilast) on gut LPMC TNFα and MMP-3 production in patients with IBD. METHODS Gut LPMCs and myofibroblasts were isolated from patients with IBD, and cultured with thalidomide, CC-4047, CC-5013, and CC-10004. MMP-3 and TIMP-1 levels were determined by western blotting and real-time PCR, and TNF-α levels by ELISA. RESULTS CC-10004 significantly reduced both TNF-α production and MMP-3 production by cultured LPMCs. Thalidomide and CC-4047 and CC-5013 had no significant effect on the production of TNF-α or MMP-3 by LPMCs. CONCLUSION These results provides a mechanistic rationale for both the failure of lenalidomide (CC-5013) in a recent randomised controlled trial in Crohns disease, and for the evaluation of CC-10004 as a novel oral therapy in the treatment of CD and UC.

P053 Interleukin-17A homodimer reduces pro-inflammatory cytokine production by inflammatory bowel disease mucosa cultured ex vivo

depends on the cell type, where it is produced and the time and location of release. IL-10 distribution in the intestine is not well known due to the methodological difficulty of cytokine staining in human biopsies. Aims: To evaluate the distribution and intensity of production of IL-10: (1) in healthy and inflamed intestine and (2) in Crohn’s disease (CD) in relation to the corticosteroid response. Methods: 26 patients with intestinal inflammation [16 Crohn’s disease (CD), 6 ulcerative colitis (UC) and 4 infectious colitis (IC)] and 10 healthy controls were included. Based on the steroid response, CD patients were classified as responders (steroid sensitive) and non-responders (steroid dependent and refractory). Samples in healthy controls were taken from ileum, left and right colon, whereas in patients samples were taken from inflamed areas. The protein expression of IL-10 was evaluated by immunofluorescence and the images obtained were processed with the ImageJ software [area (A) and mean fluorescence intensity (FI)]. We evaluated the distribution patterns and the cell type morphology. Nonparametric tests were used to compare IL-10 values between groups. Results: No significant differences in the IL-10 production were found between ileum, right and left colon in the healthy intestine (A: p = 0.589; IF: p = 0.513). No significant differences were neither found between controls and the different conditions with intestinal inflammation (A: p = 0.869; IF: p = 0.366). In CD there were no significant differences between steroid responders and non-responders (A: p = 0.913; IF: p = 0.913) In both healthy and inflamed intestine the IL-10 was distributed making a subepithelial band or making cell clusters predominantly of dendritic cells in the lamina propria. No differences in the IL-10 distribution pattern were found depending on the location in the healthy intestine (ileum or colon), nor depending on the steroid response in CD. Conclusions: The good IL-10 staining achieved by immunofluorescence reveals that this anti-inflammatory citokine is always present in along the intestine and regardless of health or disease status. The IL-10 disposition as a subepithelial band suggests an important role in defense against luminal antigens.

P073 Ex vivo activation of triggering receptor expressed on myeloid cells 1 enhances the production of pro-inflammatory cytokines by inflammatory bowel disease mucosa

BACKGROUND AND AIMS. Intestinal macrophages play a central role in the pathogenesis of inflammatory bowel disease (IBD). The phenotype of mucosal macrophages changes dramatically at the site of inflammation, where high numbers of CD33+CD68+ macrophages overexpress toll-like receptors, CD14 and triggering receptor expressed on myeloid cells 1 (TREM-1). TREM-1 is expressed on the surface of neutrophils and macrophages and contributes to the amplification of inflammatory responses by enhancing degranulation and secretion of pro-inflammatory mediators. After comparing the percentage of TREM-1-expressing macrophages in IBD versus control mucosa, we explored the ex vivo effects of an activating anti-TREM-1 antibody on the intestinal immune response in IBD. METHODS. Lamina propria mononuclear cells (LPMCs) were isolated from the inflamed colon of 11 IBD patients (5 ulcerative colitis and 6 Crohns disease), and from normal colon of 8 control subjects, and the expression of CD68, CD33 and TREM-1 was analysed by flow cytometry. TREM1 mRNA expression in CD33+ LPMCs was evaluated by qRT-PCR. IBD biopsies from inflamed mucosa were cultured ex vivo with an activating anti-TREM-1 antibody or its control isotype, and the production of pro-inflammatory cytokines, namely interleukin (IL)1beta, IL-6 and IL-8, was determined by ELISA. RESULTS. The percentage of mucosal CD33+CD68+ macrophages was significantly (p,0.05) higher both in Crohns disease and ulcerative colitis (17%±1.4 and 15%±7.6, respectively) in comparison to control subjects (6.0%±1.0). TREM-1 expression by mucosal macrophages was significantly (p,0.05) higher both in ulcerative colitis and in Crohns disease (6.4%±0.2 and 4.4%±0.6) than in control subjects (0.8%±0.1). TREM-1 transcripts were significantly (p,0.05) higher in CD33+ LPMCs from inflamed IBD compared to control subjects. TREM-1 activation significantly (p,0.01) increased IL-1beta, IL-6 and IL-8 production by IBD biopsies cultured ex vivo. CONCLUSIONS. TREM-1 is overexpressed on macrophages infiltrating inflamed IBD mucosa, and its activation amplifies the production of pro-inflammatory cytokines. Further studies using chromatin immunoprecipitation assays are underway in order to establish whether TREM-1 overexpression in IBD may derive from epigenetic changes.

P037 Is ulcerative colitis an atypical Th2-mediated disease characterised by excess production of interleukin-13?

Reference(s) [1] Atreya, R.; Neurath, M. F. (2005), Involvement of IL-6 in the pathogenesis of inflammatory bowel disease and colon cancer, Clin Rev Allerg Immu [2] Galvez-Llompart, M.; Recio, M. C.; Garcia-Domenech, R. (2011), Topological virtual screening: a way to find new compounds active in ulcerative colitis by inhibiting NF-kappaB., Mol. Divers [3] Recio, M. C.; Galvez-Llompart, M.; Zanni, R; GarciaDomenech, R. (2011), New Inhibitors of Il-6 Production In Caco-2 Cells Through Molecular Topology Methodology, XXXIII Congreso de la Sociedad Espanola de Farmacologia, Malaga (Spain).

P036 Involvement of gut macrophages in the fibrogenic process in Crohn's disease

Reference(s) [1] Atreya, R.; Neurath, M. F. (2005), Involvement of IL-6 in the pathogenesis of inflammatory bowel disease and colon cancer, Clin Rev Allerg Immu [2] Galvez-Llompart, M.; Recio, M. C.; Garcia-Domenech, R. (2011), Topological virtual screening: a way to find new compounds active in ulcerative colitis by inhibiting NF-kappaB., Mol. Divers [3] Recio, M. C.; Galvez-Llompart, M.; Zanni, R; GarciaDomenech, R. (2011), New Inhibitors of Il-6 Production In Caco-2 Cells Through Molecular Topology Methodology, XXXIII Congreso de la Sociedad Espanola de Farmacologia, Malaga (Spain).

P294 - Control of interleukin-17 production in inflammatory bowel disease

Loss of an immunological tolerance towards non-pathogenic luminal antigens is a key element in the pathogenesis of inflammatory bowel disease. Regulatory T cells (Tregs) are crucially involved in the induction and maintenance of this tolerance. Mice kept under germ-free conditions fail to induce an immunological tolerance in the gut. Intestinal epithelial cells (IECs) stimulate CD4+ Tregs dependent on MHC II. The corresponding MHC II-related processing of luminal antigens was shown to occur in multivesicular bodies (MVB). We hypothesised that the lack of tolerance induction in germfree mice might rely on defects in the MHC II-associated antigen presentation by IECs. To unravel the basic processes of the immunological homeostasis in the gut, we studied the influence of the bacterial flora on trafficking of luminal antigens into MHC II-enriched compartments responsible for antigen processing and presentation. NMRI mice were kept germ-free or conventionally colonised. Bacteria were specified in feces alliquots using 16S rRNA analysis. The results were correlated with the RDP II data base. The endocytic route of luminally applied ovalbumin (OVA) in IECs and associations with the MHC II pathway were visualised using fluorescenceand electron-microscopy. Colonised mice revealed a physiological murine flora without detection of any pathogen. IECs of the jejunum, ileum and colon lacked expression of MHC II and invariant chain (Ii) in germ-free mice. After colonisation both molecules were expressed in IECs of the jejunum and ileum, but not within the colon. While MHC II was seen within the endocytic pathway and at the basolateral membranes, Ii expression was restricted to the endocytic pathway. The majority of both antigens concentrated in MVB. In germ-free mice luminally applied OVA was only faintly detected within compartments of the endocytic tract after periods of up to two hours. In contrast colonised mice showed a significant increased uptake of OVA throughout the endocytic route (p = 0.001). Of note OVA accumulated within MHC II-enriched MVB in these mice. The bacterial colonisation of the gut seems to be essential for the MHC II-dependent antigen presentation by IECs. The physiological flora is responsible for the constitutive expression of MHC II and related molecules in IECs and a sufficient antigen delivery in processing-relevant compartments. Our data indicate that defects in the MHC II-associated antigen handling in IECs might contribute to the failure of germ-free mice in tolerance induction and support a regulatory function of these cells in the intestinal homeostasis. P294 Control of interleukin-17 production in inflammatory bowel disease L. Rovedatti1 *, A. Di Sabatino1, T. Kudo2, M. Sarra3, D. Rampton4, C.H. Knowles5, N. Sengupta5, G. Monteleone3, G.R. Corazza1, T.T. MacDonald2. 1First Department of Medicine, S. Matteo Hospital, University of Pavia, Pavia, Italy, 2Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, United Kingdom, 3Department of Internal Medicine, University Tor Vergata, Rome, Italy, 4Centre for Gastroenterology, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, United Kingdom, 5Centre for Academic Surgery, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London, United Kingdom