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Background: We previously demonstrated that in inflammatory bowel disease (IBD) there is enhanced production of interleukin (IL)-21, a cytokine that activates multiple pathways that sustain mucosal inflammation. However, the phenotype of IL-21-producing cells in IBD, and the cytokine(s) they coproduce, is not known. We here characterized the cell source of IL-21 and determined which factors regulate IL-21 in the human gut. Methods: Cytokines were analyzed in CD4+ T intestinal lamina propria lymphocytes (T-LPL) isolated from IBD patients and controls by flow cytometry. Moreover, IL-21 was evaluated in mucosal T follicular cells (TFH). To assess the involvement of IL-12 and IL-23 in the production of IL-21, T-LPL were activated in the presence or absence of IL-12 or IL-23. Results: The proportion of IL-21-producing CD4+ T-LPL was increased in IBD compared to controls. The majority of IL-21-producing T-LPL coexpressed interferon (IFN)-γ, and to a lesser extent IL-4 or IL-17A. Activation of CD4+ T-LPL with IL-12 but not IL-23 enhanced the fraction of cells coexpressing IL-21 and IFN-γ. TFH cells in LPL were identified by CXCR5 expression and expressed IL-21 both in IBD and controls; however, the fraction of IL-21-positive TFH cells was higher in Crohn's disease than in ulcerative colitis and controls. Treatment of CD4+ T-LPL with IL-12 enhanced the frequency of CXCR5+ IL-21-producing TFH cells. Conclusions: These findings indicate that in IBD IL-21 is mostly produced by CD4+ T-LPL coexpressing IFN-γ, reinforcing the concept that distinct subsets of T cells can produce IL-21. Inflamm Bowel Dis 2010

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 Crohn's disease, and for the evaluation of CC-10004 as a novel oral therapy in the treatment of CD and UC.

Background: Intracellular kinase activation plays a key role in inflammation and kinase inhibitors have been proposed as potential therapy for chronic inflammatory disorders such as ulcerative colitis. Selective kinase inhibitors have proved disappointing, particularly in the treatment of rheumatoid

If we understand pathological processess within the alimentary tract, it is apparent that the fundamental aspects of microbe-host interactions need to be examined in greater detail. Pathogenic bacteria have evolved strategies to alter and subvert the function of T cels and phagocytes in the gut wall, and exploiting these molecules may lead to new treatments for chronic inflammatory bowel diseases. The adaptation of microbes to their host must involve microbe-mediated interference of the host innate immune response. The recent demonstration that nonpathogenic E. coli have a beneficial effect in ulcerative colitis further supports the notion that normal flora may alter the expression of the innate immune receptors or recognize alternative receptors compared with pathogenic variants. Such differences may conceivably lead to beneficial and protective alterations to the host through cytokine and antimicrobial peptide expression. Perhaps the contact point between microbes and host cells lies with the pattern-recognition receptors such as TLRs. However, although much light has been shed on the downstream consequences of TLR activation, many more questions remain unsolved. For example, little is known about the expression profiles of the different TLRs throughout the gastrointestinal tract. Additionally, ambiguities remain over the natural ligands for TLRs. The discovery that the Drosophila Toll receptor acts downstream of the pathogen recognition event suggests that there are many more twists and turns to be revealed in the story of host-microbe interactions in the gastrointestinal tract.

In the intestine large numbers of bacteria and their products are separated by a single epithelial layer from resident inflammatory cells (macrophages and lymphocytes). Many of these bacterial products, such as lipopolysaccharides and peptidoglycans, are potent stimulators of free radical and inflammatory cytokine production by macrophages. This can occur in vivo in response to mucosal invasion by enteropathogenic bacteria or because of inappropriate activation of these cells, as in chronic inflammatory bowel disease. In this review we present evidence for production of cytokines in normal intestine and in intestinal inflammatory conditions. The adverse effects of cytokine production upon intestinal homeostasis, in particular disruption of epithelial integrity and prothrombotic changes in the vascular endothelium, are also discussed.

SUMMARY In this study we examined the distribution of the adhesion molecules ICAM-1, VCAM-1 and E-selectin in human fetal intestine, to determine whether they may have a role in the development of gut-associated lymphoid tissue. Secondly, we studied the tempo of induction of these molecules after T cell activation in explants of human fetal intestine cultured in vitro. In the fetus from 11 to 20 weeks gestation, endothelial expression of ICAM-1 and diffuse staining of VCAM-1 was observed in the lamina propria. In contrast, there was intense expression of ICAM-1 and VCAM-1 in the developing Peyer's patches, suggesting that these molecules may be involved in the accumulation or organization of lymphoid tissue in the gut. After T cell activation in fetal intestinal explants, the expression of ICAM-1 and VCAM-1 was increased on most endothelial cells, leucocytes, and stromal cells in the lamina propria. Expression was maintained for at least 4 days. In contrast, the induction of E-selectin was rapid, and the expression was transient, despite the continuing presence of activated T cells and macrophages. This suggests that other factors are required to prevent the down-regulation of E-selectin to maintain the sustained expression sometimes observed in vivo.

SUMMARY The spot-ELISA technique has been used to enumerate the frequency of cells secreting tumor necrosis factor-alpha (TNF-α) and interferon-γ (IFN-γ), isolated from biopsies of normal intestine and from biopsies of children with inflammatory bowel disease. TNF-α production was undetectable in six out of 12 biopsies from normal intestine and in the other six biopsies it ranged from 60 to 580 TNF-α-secreting cells/106 isolated intestinal cells. In contrast, cells isolated from biopsies of children with Crohn's disease (n= 9) all showed elevated frequencies of TNF-á-secreting cells (500–12 000 secreting cells/106 cells). In ulcerative colitis, four out of eight children had increased production of TNF-α and in children with indeterminate colitis two out of three had elevated levels. There was no correlation between plasma TNF-α levels and the number of intestinal cells secreting TNF-α. In controls and all groups of patients IFN-γ-secreting cells were uncommon. These results suggest that TNF-α is an important mediator of inflammation in the human gut, and, furthermore, may play a role in the growth failure frequently seen in children with inflammatory bowel disease.

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.

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).

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).

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