Sa1777 Ex Vivo Activation of Triggering Receptor Expressed on Myeloid Cells 1 Enhances the Production of PRO-Inflammatory Cytokines by Inflammatory Bowel Disease Mucosa

site of inflammation in Crohn`s disease (CD) has been described. However, the functional consequences of their presence remain controversial. Here, we show that PMN in mucosa from CD patients acquire APC characteristics. Materials and methods: Freshly isolated peripheral blood PMN from healthy volunteers (HC) and CD patients were cultured for 3 days in the presence of cytokines (GMCSF 50U/ml, INF γ 100U/ml and IL-4 3ng/ml). Expression of CD80, CD86, MHC II on CD66b+ PMN were measured by flowcytometry in peripheral blood and single cell suspensions from biopsies. PMN expression of MHC-II at the site of inflammation was determined by double labelling of biopsies taken from small intestine and colon both at the inflammed and non-inflammed sites. Results: PMN present in the peripheral blood from CD patients and HCs did not express the co-receptors for antigen presentation. However, mucosal PMN from fresh CD biopsies did express APC coreceptors, both at site of inflammation and in non-inflamed regions (n=5, 13±2 % of PMN vs 15±6%). In contrast, mucosal PMN from healthy control biopsies did not express CD80, CD86 or MHC II. As a confirmation of the flowcytometric data, biopsies from CD patients and HCs were stained for MHC-II in conjunction with the PMN marker CD66b. Double positive cells were found only in active CD colonic and small intestinal biopsies. To investigate whether the propensity to dedifferentiate was intrinsically enhanced in CD PMN, peripheral blood PMN were cultured in the presence of a cytokine cocktail, inducing dedifferentiation and expression of CD80, CD86, MHC II. However, similar percentages of PMN expressed APC markers in CD (9.6±6 %, n= 8) and HC (15±10.6%, n=11) in vitro. Conclusion: We show for a first time that a subset of PMN in active CD patients expresses antigen-presenting co-receptors by flowcytometry as well as by immunofluorescence. Induction of redifferentiation of PMN from CD and HC in vitro is not different, indicating that the intrinsic capacity of PMN to dedifferentiate is not affected in PMN. It is likely that the local presence of proinflammatory cytokines induces this dedifferentiation of PMN within the mucosa of CD patients. This may serve to enhance presentation of bacterial antigens to T cells, thereby adding fuel to the already over-activated T cell response in CD.

AGA Abstracts

Sa1777 Ex Vivo Activation of Triggering Receptor Expressed on Myeloid Cells 1 Enhances the Production of PRO-Inflammatory Cytokines by Inflammatory Bowel Disease Mucosa Paolo Biancheri, Francesca Ammoscato, Antonio Di Sabatino, Renata Curciarello, Laurens Kruidenier, Gino R. Corazza, Thomas T. MacDonald 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 Crohn's 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 Crohn's 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 Crohn's 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.

Sa1780 Comprehensive T Helper Cell Profiling Reveals Concomitant Upregulation of Regulatory T Cells and TH17 Cells in Active Inflammatory Bowel Disease Bertram Bengsch, Anna-Maria Globig, Nadine Hennecke, Maximilian Seidl, Hubert E. Blum, Robert Thimme Background: Skewed T helper cell responses have been implicated in the pathogenesis of inflammatory bowel disease (IBD). In seminal studies, a strong TH1 response has been linked to Crohn's disease (CD) and a strong TH2 response was associated with ulcerative colitis (UC). Recently, several studies could identify prominent TH17 responses in patients with active disease that correlated with a decreased regulatory T cell (Treg) response. However, there is limited information about the exact contribution of these T helper cell subsets to inflammation in active IBD. Thus, we assessed the individual contribution of different T helper cell subsets by performing a comprehensive analysis of TH1, TH2, TH17 and Treg cells in patients with CD (n=40) and UC (n=32) undergoing colonoscopy or surgical resection and compared them to healthy controls (n=40) undergoing routine colonoscopy. Methods: Following isolation from gut tissue or the peripheral blood, T cells were analyzed for the production of the cytokines IFN- γ, TNF and IL-17 as well as the expression of CD25, CD26, CD27, CD28, CD45RA, CD103, CD127, CCR7, CRTH2 and FoxP3. After evaluation of several methods to assess the intensity of inflammation, biopsies were further evaluated for inflammatory activity by a single pathologist in a blinded manner. Results: Overall, peripheral lymphocyte differentiation did not correlate with differentiation of intestinal T cells. Compared to the peripheral blood, specialized T helper subsets were enriched in the intestinal mucosa and naive T helper cells were decreased. By analyzing the intestinal T cell pool, we observed similar frequencies of TH1 and TH2 cells in patients with CD and UC and healthy controls. In contrast, TH17 responses were enriched in the intestinal tissue of patients with IBD. Stronger histological inflammation correlated with stronger TH17 responses. Importantly, we observed a concomitant up-regulation of Tregs with TH17 cells in inflamed tissues. Conclusion: These results argue against a clear link of TH1 and TH2 cells with CD and UC, respectively. They suggest that TH17 cells are strongly involved in the pathological inflammatory response in patients with IBD. Importantly, the concomitant upregulation of Tregs despite ongoing inflammation indicates an impaired functional response of regulatory T cells.

Sa1778 Genetically-Determined Cytokine Programs of Human Natural Killer (NK) Cells Uncover a Novel Mechanism of NK Cells and Kir Genes in Crohn's Disease Susceptibility Lin Lin, Chao Ma, Bo Wei, Raja Rajalingam, Susy Yusung, Elizabeth A. Trachtenberg, Stephan R. Targan, Dermot P. McGovern, Jonathan Braun Background: Mucosal innate lymphocytes have emerged as an important new factor in IBD pathogenesis, but the role of NK cell subpopulation in IBD is poorly understood. A recent genetic study has identified KIR2DL2/3, a natural killer (NK) receptor allele, as a risk factor for CD in the genetic context of its cognate ligand HLA-C1 (Hollenbach JA. 2009 Immunogenetics. 61: 663). However, the biologic mechanism of this genetic contribution is unknown. NK cells are mainly known for their cytolytic function, but recent work indicates that strong NK receptor/MHC1 interactions can induce a program of NK differentiation ("licensing") that includes augmented NK cytokine production. We therefore tested the hypothesis that human NK licensing by KIR2DL2/3 and HLA-C1 induces an NK cytokine program that modifies the threshold of pro-inflammatory T cell activation. Methods: A cohort of 80 subjects (30 CD and 50 controls) was genotyped for HLA and KIR, and blood NK and CD4+ T cells were isolated for study. Licensed and unlicensed NK cells were defined based on combinatorial genetics and expression of KIR genes by flow cytometry. The NK cytokine program was determined at the single-cell level using single cell barcode chip (SCBC). Co-culture of CD4+ T cells with NK cells or supernatants were conducted to test for costimulation of antigenic CD4+ T cell activation and TH1 or TH17 cell subset polarization. Neutralizing antibody and add-back experiments validated candidate NK cytokines involved in these interactions. Results: Licensed versus non-licensed NK cells had strikingly distinct cytokine programs: licensed NK cells were distinguished by rich poly-cytokine expression at high secretion levels and a pro-inflammatory mode; non-licensed NK cells produced few cytokines at low secretion levels and a regulatory and tissue remodeling mode. Licensed NK cells had a much higher capacity for co-stimulatory augmentation of antigenic CD4+ T cell activation. This was attributable to NK cell cytokines rather than NK-T cell-cell interaction, and blocking and add-back experiments identified the key molecules as IL-6, TNF- α, and IFN-γ. CD and control subjects displayed the findings, indicating that the effect of licensing on NK-T interaction is a genetic process not secondary to disease state. Conclusions: NK licensing mediated by KIR2DL2/3 and HLA-C1 induces a novel and striking cytokine program of NK cells that reduces the threshold to form pro-inflammatory CD4+ T cells, and therefore provides a predisposing biologic mechanism for this genetic trait and CD. These findings reveal a new biologic paradigm to understand KIR-associated susceptibility to CD and other chronic inflammatory syndromes.

Sa1781 Thiopurine Analogues for Optimized RAC1/Vav1 Blockade in Inflammatory Bowel Diseases Imke Atreya, Alexandra Diall, Raja Atreya, Stefanie Zenker, Christian Henninger, Gerhard Fritz, Radovan Dvorsky, Mathias Gruen, Ute Hofmann, Elke Schaeffeler, Ilse Daehn, Rocio Lopez-Posadas, Matthias Schwab, Markus F. Neurath Introduction: 6-mercaptopurine represents an effective immunosuppressive agent for therapy of autoimmune and chronic inflammatory diseases such as inflammatory bowel diseases. This drug induces T cell apoptosis for immunosuppression via blockade of the Vav1/Rac1 interaction. Nevertheless, the use of 6-mercaptopurine is limited by the delayed onset of action and potential severe side effects. To overcome these limitations, the here described project aims at the development of a new and improved class of thiopurine-based immunosuppressive agents. Methods: Based on the identification of 6-thio-GTP as the key active metabolite of 6-mercaptopurine, 44 chemically modified 6-thio-GTP analogues were designed by steric modelling. New 6-thio-GTP analogues were afterwards tested on isolated human T cells for their pro-apoptotic capacity. The potential clinical applicability of the best candidates was evaluated by in vitro toxicity tests and the proapoptotic capacity of our lead compound was confirmed by in vitro apoptosis assays in lamina propria mononuclear cells of IBD patients. Results: Out of all tested 6-thio-GTP analogues, the lead compound B-0N was selected by its superior capacity to mediate earlier and stronger induction of T cell apoptosis than 6-mercaptopurine. Accordingly, B-0N treatment of stimulated lamina propria mononuclear cells isolated from inflamed colon tissue of IBD patients resulted in a successful and significant induction of apoptosis. The optimized immunosuppressive function of B0N was further underlined by the fact that B-0N led to a significantly reduced production

Sa1779 Adding Fuel to the Fire - Neutrophils As Antigen Presenting Cells in Crohn`S Disease Rajesh somasundaram, Christien J. van der Woude, Maikel P. Peppelenbosch, Gwenny Fuhler Introduction: Traditionally, neutrophils (PMN) are considered to be the first line of defence against bacterial infection. However, it is becoming clear that they may also participate as antigen-presenting cells (APCs), as observed in inflammatory diseases like rheumatoid arthritis and bacterial infections. During inflammation, PMN are exposed to proinflammatory cytokines which not only influence PMN survival, but upon prolonged exposure can also lead to gain of new function - antigen presentation to T cells. Neutrophil influx at the

AGA Abstracts

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