M1677a The Ubiquitin-Editing Enzyme A20 Restricts TCR Induced NFkB and T Cell Activation

AGA Abstracts

indicate that the stimulations of α7nAChRs on the DCs in the colon induce anti-inflammatory action by an inhibitory regulation of enhanced Th1 and Th2 immune responses in the colitis model. Our findings suggest that DCs, antigen-presenting cells in lamina propria of colon possess the inhibitory α7nAChR against the inflammation to prevent over-activation of immune responses in the mucosal immune system of the colon. Our results may contribute to the development of therapeutic medicines for modifying the cholinergic anti-inflammatory pathway to treat various enteric inflammatory diseases.

vs M-SAMP (N=20/12; 10.8±1.0 vs 6.9±0.8, P<0.01); this trend continued until 20 wks of age (N=17/18; 15.8±0.7 vs 12.7±0.8, P<0.01), with no inflammation in control AKR mice. FACS data revealed a significantly higher population of nTregs (CD25+FoxP3hi) in M- vs F-SAMP (N=12; 10±0.7 vs 6.2±0.4%, P<0.01), while inducible Tregs (CD25+FoxP3lo) were greater in F- vs M-SAMP (N=12; 7.0±0.59 vs 3.7±0.56% P<0.01). Presence of the cell surface marker, FR4 (folate receptor 4), suggested a significant increase in Tregs with suppressive function in M- vs F-SAMP (M/F, N=12; 6.6±0.68 vs 5.2±0.6%, P<0.05), while functional assays on co-cultured, magnetically-sorted CD4+CD25- and CD4+CD25+ MLN cells (2:1 ratio) confirmed these findings, indicating greater suppressive activity of Tregs from MSAMP, with the lowest percentage of proliferating CD4+CD25- cells, compared to F-SAMP (9.0±2.7 vs 18.2±3.5%), as well as M- and F-AKR controls (10.2±4.4 and 14.7±5.7%, respectively). Finally, stimulation of sorted MLN Tregs with an estrogen receptor β-specific agonist showed significant upregulation of both FoxP3 (N=7/6; 15.3±0.7 vs 4.9±1.9%, P<0.01) and TGFβ (N=7/6; 548.8±54.4 vs 320.5±12.7 pg/ml, P<0.01) in M- vs F-SAMP, indicating estrogen-dependent regulation of Treg function. Conclusion: Taken together, these data suggest gender-dependent differences of ileitis in SAMP mice may be due to intrinsic disparity in the phenotypic and functional characteristics of Treg populations, which are influenced by sex hormone levels, and may ultimately affect immune tolerance and the onset and severity of chronic intestinal inflammation such as that observed in CD.

M1677 Imprinting of Lymphocyte Homing to Skin and Gut By Human Tissue DC Elizabeth R. Mann, Hafid O. Al-Hassi, Susan K. Clark, Neil E. McCarthy, Stella A. Cochrane, Ailsa Hart, Andrew J. Stagg, Stella C. Knight INTRODUCTION: Dendritic cells (DC) play a crucial role in the induction of primary immune responses via their ability to prime naïve T-cells. Upon DC activation, T-cells change their pattern of adhesion receptors and acquire the capacity to migrate to the peripheral tissues. However, mechanisms by which DC imprint tissue homing phenotypes on T-cells are not fully understood. DC imprinting pathways may be dysregulated in T-cell dominated inflammatory disease such as inflammatory bowel disease (IBD). METHODS: We analysed expression of tissue homing molecules on human DC and T-cells in the peripheral blood and tissues, both pre and post allogeneic co-culture. Rectal biopsies and skin samples were obtained from healthy volunteers to obtain “walkout” cells. Mononuclear cells were gradientseparated from human whole blood and depleted of HLA-DR+, CD14+ and CD19+ antigenpresenting cells prior to 5 day culture with enriched blood DC (LDC) or mucosal “walkout” cells. Cells were labelled with monoclonal antibodies and analysed by flow cytometry. RESULTS: The majority of blood DC co-expressed gut homing marker β7 integrin and skin homing marker CLA. In contrast, T-cells predominantly expressed either β7 (mainly CD45RO- cells) or CLA (mainly CD45RO+ cells), with the majority being β7+ only. DC and T-cells from the skin only expressed CLA, but DC and T-cells from the gut only expressed β7. However, In Vitro stimulation of T cells with blood LDC induced co-expression of CLA with β7, while stimulation with mucosal DC induced β7 only. CONCLUSIONS: In the steady state, the majority of blood T-cells appear to be re-circulating to the gut and exhibit a “naïve” phenotype, while a smaller “memory” cell population exhibit a skin homing profile. Blood DC and T-cells stimulated with blood DC co-express skin and gut homing receptors, enabling migration to diverse tissues in early stages of an immune response. However, T-cells stimulated with gut DC are capable of migration to the gut only. Dysregulation of these imprinting pathways is likely to play a pivotal role in inflammatory disease and may contribute to extra-intestinal skin manifestations that sometimes occur alongside IBD. Further work will determine how these pathways should be targeted for therapeutics in inflammatory disease.

M1675 TGFβ and IFNα/β Play Major Roles in Induction of Antigen-Independet Regulatory T-Cells Mediated By CpG Motifs of Bacterial DNA Andre Bleich, Lydia M. Janus, Anna Smoczek, Hans J. Hedrich, Stefan Fichtner-Feigl, Werner Falk, Claudia Hofmann, Florian Obermeier Background and Aims: Prophylactic treatment of mice with CpG motifs of bacterial DNA protects from experimental IBD, at least partly via induction of inhibitory T-cells. We have previously shown in a cell transfer model using germ-free mice that this CpG-mediated tolerance is even induced without pre-existence of bacterial flora, indicating that CpG-ODN induced regulatory T-cells are not bacterial antigen specific. Aim of this study was to identify factors that mediate the CpG-ODN dependent induction of protective properties in T-cells under germ-free conditions. Material & Methods: Germ-free BALB/c and Il10-/- mice were treated for three weeks (Il10-/- only one week) twice weekly with 10 μg CpG-oligodeoxynucleotides (ODN), control GpG-ODN, or PBS (sham-treated). In parallel with each ODNtreatment, antibodies directed against type-I-interferons (100 μg each time; rat anti-MulFNα [clone 4E-A1] and anti-MulFN-β [clone 7F-D3] Mabs) or against TGFβ (1 mg each time) or control rat immunoglobulin (IgG) were applicated by i.p. route. Germ-free mice served as donors for splenic CD4+CD62L+ cells that were transferred by i.p. injection into CB17Prkdcscid (SCID) mice maintained under SPF conditions. After 8 weeks, colitis development was evaluated histologically and cytokine production of mesenteric lymph node (MLN) cells of recipients was determined by ELISA. Results: Histological analysis of recipient mice revealed that control rat IgG application did not change the protective effect of CpGODN treatment, while both anti-IFNα/β and anti-TGFβ treatment resulted in a partial but significant abrogation of the CpG-ODN induced protection. Histological data were corroborated by increased TNF and decreased IL10 secretion of MLN cells isolated from recipient mice. These CpG-ODN mediated effects were slightly more effectively blocked by neutralizing IFNα/β than TGFβ; IFNγ and IL17 secretion were only affected by anti-IFNα/ β but not anti-TGFβ treatment. A slight but not significant protection was observed in SCID recipients that received cells from CpG-ODN treated germ-free Il10-/- donor mice. Conclusions: TGFβ likely plays a considerable role during the CpG-ODN mediated induction of regulatory T-cells in germ-free mice. Type-I-interferons, that were identified previously as a mediator of CpG-ODN protection likely by effects on innate immunity, are also protective in the transfer model. Although IL10 independent mechanisms play a role in CpG-ODN protection as shown previously, this cytokine likely is important for the effector mechanism of CpG-ODN induced regulatory T-cells.

M1677a The Ubiquitin-Editing Enzyme A20 Restricts TCR Induced NFkB and T Cell Activation Shigeru Oshima, Rita M. Tavares, Emre Turer, Regina-Celeste Ahmad, Averil I. Ma Background & Aims: Our earlier studies of A20 revealed that A20 deficient mice develop severe spontaneous inflammation and colitis. However, A20 is expressed in many cell types, and may restrict NFkB signaling in these cells. One key cell type is T cells. Stimulation of T cell receptor (TCR) is required for activation of T cells, so regulating TCR signals controls whether T cells cause intestinal inflammation. We investigated whether A20 regulates TCR signals and T lymphocyte function in the intestine. Methods: To investigate A20's role in specifically regulating T cells and colitis, we used recombineering and gene targeting to generate mice bearing a mutant allele of the A20 gene which contains LoxP recombination sites (“A20flox” mice). A20flox mice were then bred to either Lck-Cre or CD4-Cre transgenic mice to establish complementary models of mice lacking A20 expression specifically in T cells. We also interbred A20flox mice with Rosa-ER/Cre mice which express an estrogen receptor-Cre fusion protein that is activated by 4-hydroxy-tamoxifen (4-OH-T). Results: Lck-Cre and CD4-Cre transgenic mice contained relatively normal numbers of lymphocytes, but a significant proportion of T cells were spontaneously activated by flow cytometric markers. Thus, A20 expression in T cells prevents their spontaneous activation. As T cells in Lck-Cre A20flox and CD4-Cre A20flox mice were spontaneously activated, measuring responses in naïve A20-/- T cells remained challenging. We thus developed an approach in which we could acutely delete A20 in primary T cells and then test their functions. The resulting Rosa-ER/Cre A20flox mice possess normal T cells. Acute deletion of A20 from mature T cells with 4-OH-T and adoptive transfer into RAG-1 deficient mice led to rapid (<3 wks) weight loss and colitis compared to control T cells. This inflammation was associated with elevated levels of IFNg. To understand the biochemical mechanisms by which A20 may restrict T cell activation and colitis In Vivo, we studied T cell activation In Vitro. We found that A20 deficient T cells exhibit prolonged NFkB signaling and increased IL-2 production after T cell receptor stimulation. Conclusions: Thus, A20 restricts TCR induced NFkB signaling and T cell activation, thereby preventing colitis.

M1676 Cholinergic Anti-Inflammatory Effect Through Activation of Alpha7 Nicotinic Acetylcholine Receptor On CD11c+ Lamina Propria Dendritic Cells in Oxazolone-Induced Colitis Takeshi Yamamoto, Minako Yoshida, Kanae Fujiwara, Makoto Kadowaki Some clinical trials and epidemiologic studies show that smoking and nicotine patch improve symptoms of ulcerative colitis (UC). In addition, it has been demonstrated that vagus nerves through activation of α7 nicotinic acetylcholine receptor (nAChR) on the immune cells take part in the inhibitory regulation of proinflammatory cytokines. We have previously reported that subcutaneous administration of nicotine ameliorates oxazolone (OXZ)-induced colitis, which are reversed by pretreatment of non-selective nAChR antagonist hexamethonium or α7nAChR antagonist methyllycaconitine. Furthermore, in the middle colon of OXZ colitis, the treatment of nicotine suppresses the over-expressions of T helper cell type 1 (Th1) as well as Th2 cytokine mRNAs. Therefore, the purpose of the present study is to determine which immune cells express α7nAChRs in the lamina propria of the colon. Methods: OXZ colitis was developed in BALB/c mice. Unfixed cryosections of colon from the mice were stained with FITC-conjugated α-bungarotoxin (FITC-α-BTx) to label α7nAChRs. Lamina propria mononuclear cells (LPMCs) were collected from colon of OXZ colitis mice. First, LPMCs were separated into CD3e, CD45R/B220, or CD11c positive rich fractions using the magnetic cell separation system (BD biosciences). Second, LPMCs were separated into dendritic cells (DCs) rich fraction using DCs enrichment set (BD biosciences). Each separated fraction of LPMCs was analyzed by flow cytometry and then by real-time PCR. Result: Morphological studies with FITC-α-BTx demonstrated that FITC-α-BTx labelled α7nAChRs were expressed in the mucosa of OXZ colitis mice colons and were up-regulated following the onset of OXZ colitis. In the separated CD3e, CD45R/B220, or CD11c positive rich fractions of LPMCs of OXZ colitis mice colons, quantification of α7nAChR mRNA expression with real-time PCR revealed that α7nAChR mRNA were mainly expressed in CD11c positive rich fraction. Furthermore, α7nAChR mRNA expression in the DCs of LPMCs was confirmed in the cells separated from LPMCs using DCs enrichment set. Conclusion: Our results

AGA Abstracts

M1678 EPEC Decreases Surface NHE3 Levels Through Ezrin Dephosphorylation Kim Hodges, Krishnamurthy Ramaswamy, Pradeep K. Dudeja, Gail A. Hecht Sodium hydrogen exchanger 3 (NHE3) is bound to the actin cytoskeleton through two independent interactions with the scaffolding protein ezrin one of which is mediated by NHE regulatory factors or NHERFs. Phosphorylation of ezrin on threonine 567 blocks an intermolecular interaction allowing the C-terminus of ezrin to bind to actin and the Nterminus to bind to target proteins including NHE3 and NHERF1/2. In contrast, dephosphorylation converts ezrin to a closed conformation that prevents cytoskeletal interaction. Enteropathogenic Escherichia coli (EPEC) regulate NHE3 activity through effects on ezrin. EPEC express a type III secretion system (T3SS) that spans the bacterial and host cell

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