IL-10 secretion and Foxp3 induction. PI3K signaling mediates bacteria-stimulated B cellproduction of IL-10.

Tu1959

Disruption of the Intestinal Barrier Through M1 Macrophages - Relevance forIBD?Donata Lissner, Michael Schumann, Thorsten Stroh, Lea I. Kredel, Arvind Batra, Anja A.Kuehl, Martin Zeitz, Britta Siegmund

Background Recent studies suggest a beneficial role for regulatory macrophages in inflammat-ory bowel disease (IBD). Our aim was to characterize the effect of macrophage subtypes onthe epithelial barrier. Methods Intestinal resection specimens of patients with Crohn's disease(CD) and ulcerative colitis (UC) were analyzed by immunhistochemistry and compared tohealthy controls. Peripheral blood monocytes were isolated and polarized into M1- and M2-macrophages. The influence of these cells on epithelial integrity was analyzed using resistancemeasurements on different epithelial cell lines (Caco-2, T-84, HT-29/B6). Cytokine concen-trations were determined by cytometric bead array. Results In both, IBD patients and healthycontrols, CD163+ and stabilin1+ cells were found within the lamina propria, suggesting apermanent presence of M2-macrophages in this compartment. Exlusively in inflamed gut,and here predominantly in CD patients, iNOS+ cells were found subepithelially, suggestingan accumulation of M1-macrophages and thus shifting the balance to a pro-inflammatorystate. In-vitro polarized M1-macrophages caused a profound decrease in resistance of epithe-lial cell layers compared to non-polarized or M2-macrophages, which was further aggravatedthrough stimulation with LPS. Paracellular leakage was revealed by filter staining throughderegulation of various tight junction proteins as well as induction of apoptosis in epithelialcells. High concentrations of TNFα were found in the supernatants of M1-macrophages,whereas the predominant cytokine for M2-macrophages was IL-10. Consequently, the effectof M1-macrophages on epithelial cells was partly abrogated by inhibition of TNFα throughinfliximab. Conclusion The pro-inflammatory milieu in the lamina propria of Crohn's diseasepatients is shaped by the presence of M1 macrophages. This milieu plays a critical role inmediating an increased leakiness of the epithelial barrier, a prerequisite for the maintenanceof intestinal inflammation.

Tu1960

Lamina Propria CD4+Lap+ T Cells Show In Vitro Regulatory Activity and areSelectively Increased in Active Ulcerative ColitisAntonella D'Ambrosio, Andrea Cossu, Massimo Sanchez, Annamaria Pronio, ChiaraMontesani, Mauro Di Camillo, Piero Vernia, Roberta Pica, Anna Kohn, Monica Boirivant

Background: A CD4+CD25- regulatory T cell population expressing the surface TGF-β inits latent form LAP (Latency Associated Peptide) was previously identified and proved tobe protective in the adoptive transfer colitis model (J. Immunol. 2003,170:2516). In recentstudies we demonstrated that lamina propria (LP) CD4+LAP+ T cells population appears tobe relevant to protect mice from TNBS-induced colitis (J. Immunol. 2005,174: 3237). Wealso showed, for the first time, the presence of LP CD4+LAP+ T cells in patients with IPAAfor ulcerative colitis (UC) (IBD 2008, 14: 662). Recently peripheral blood (PB) CD4+LAP+T cells from healthy subjects have been shown to inhibit the proliferation of PB CD4+LAP−T cells (J. Immunol. 2010,184;4620). More recently induced CD4+LAP+ T cells have beenreported to reduce the TNF-α production by LPS treated PBMC(peripheral blood mononu-clear cells). (Immunol. 2011, 133; 278). Aim: In the present study we investigated theprevalence and function of LP CD4+LAP+ T cells in inflammatory bowel disease (IBD)patients. Methods: Specimens from patients undergoing colonscopy or bowel resection forIBD and cancer were used as the source of LPMC. UC groups were divided according toendoscopic activity (modified Baron index -New Eng J Med 2005;352: 2499). IL-17 produc-tion, LAP and Foxp3 expression in CD3/CD4 gated cell population was assessed ex-vivoby immunofluorescence. CD4+LAP+CD25- and CD4+LAP- CD25- LP cells were FACS-sorted for functional studies. Results: In initial studies we found that LPMC from Crohn'sdisease(CD) patients showed a reduced % CD4+LAP+ T cells while LPMC from UC patientsshowed an increased proportion of CD4+LAP+ T cells when compared to controls. Insubsequent studies, we found that the majority of LP CD4+LAP+ from UC patients wereFoxp3- and that LP CD4+LAP+ CD25- sorted cells from mild UC specimens or controlswere able to inhibit the proliferation of In Vitro αCD3/28 stimulated autologous LP and PBCD4+LAP- CD25- cells. The % of CD4+LAP+ cells was significantly higher in LPMC isolatedfrom moderate to severe active UC patients when compared to mild / inactive patients whoshowed values comparable to controls (C) (10.3%±2 UC vs 3.5%±1.5 C; mean ± SEM, p=0.015). Initial characterization of these cells showed that a variable proportion (0-26%) ofLP CD4+ LAP+ gated T cells were IL-17+. The proportion of LP CD4+ LAP+ gated T cellswas significantly higher in active UC patients when compared to control patients (19.6%±1vs 3.4% ± 2.2, respectively, p=.00029). LP CD4+IL-17+cells were also increased in activepatients when compared to C. Conclusions: In humans, LP CD4+LAP+ cells show regulatoryactivity. In IBD, they are selectively increased in active UC patients where a proportion ofLP CD4+LAP+ cells show IL-17 expression.

Tu1961

Inhibition of Autotaxin-Lysophosphatidic Acid Axis Significantly AmelioratesChronic Intestinal Damage by Modulating Lymphocytes Migration to IntestinalMucosaHideaki Hozumi, Ryota Hokari, Chie Kurihara, Shingo Sato, Toshihide Ueda, MasaakiHigashiyama, Keisuke Okudaira, Yoshikiyo Okada, Chikako Watanabe, ShunsukeKomoto, Kengo Tomita, Atsushi Kawaguchi, Shigeaki Nagao, Soichiro Miura

Background: Aberrant leukocyte migration has been implicated in the pathogenesis of inflam-matory bowel diseases (IBD). Recently, lysophosphatidic acid (LPA) is reported to play acritical role in lymphocyte migration to the secondary lymphoid organization. In addition,autotaxin(ATX)/lysophospholipase D in vascular endothelium is reported to be the main

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enzyme in the production of LPA. Nevertheless, there have been no studies whether ATXis involved in the aberrant lymphocyte migration to the inflamed intestinal mucosa of IBD.We previously reported that enhanced expression of ATX mRNA was observed in the highendothelium venule-like vessels in the inflamed intestine of patients with IBD (DDW2011).In this study we investigated the possible involvement of ATX in the colonic inflammationusing two murine colitis models, and the role of ATX in lymphocyte transmigration throughinflamed vascular endothelium using transwell assay system. Method: In murine study, tissuesamples were obtained from colon of SCID mice transferred with CD4 cells of spleen, andcolon of BALB/c mouse provided with drinking water containing DSS. Degree of expressionof ATX mRNA was determined by using quantitative RT-PCR. The inhibitory effect ofbithionol (an ATX inhibitor) on aberrant lymphocytes migration to the intestinal mucosaas well as intestinal damages were evaluated. To induce high endothelial venules In Vitro,bEnd3 cell line was treated with TNF-alpha (5ng) for 3 days. Induction of MAdCAM-1 andATX was confirmed by RT-PCR. Transendothelial migration of splenocytes through highendothelial vessels was studied by using transwell assay. Inhibitory effect of ATX and BrP-LPA on the number of transmigrated splenocytes was determined. Surface expression oftransmigrated splenocytes was determined by flowcytometry. Result: In CD4 transferredSCID mice model, degree of expression of ATX mRNA gradually increased after 8 weeks ascolitis developed. In DSS mice model, degree of expression of ATX mRNA was significantlyhigher in the colonic mucosa of chronicically developed colitis than that of acute phase.Administration of bithionol significantly ameliorated both DSS-induced colitis and CD4-induced ileocolitis. In transwell assay, treatment of bithionol or a LPA inhibitor significantlydecreased transmigration of splenocytes. Conclusion: Blocking of ATX expression successfullyattenuated chronic intestinal damage possibly via inhibition of aberrant lymphocyte migrationto the inflamedmucosa ofmurine colitis. Transendothelial migration through high endothelialvessels was inhibited by ATX or a LPA inhibitor. Enhanced expression of ATX in the activemucosa suggests that autotaxin/lysophospholipase D becomes a new therapeutic target forIBD treatment.

Tu1962

Microbial Exposure During Early Life Has Persistent Effects on Tissue-Associated iNKT Cells and Their FunctionTorsten Olszak, Dingding An, Sebastian Zeissig, Miguel Pinilla Vera, Jonathan Glickman,Rebecca M. Baron, Dennis L. Kasper, Richard S. Blumberg

Background & Aim: Epidemiologic studies support the importance of exposure to microbesduring early childhood in subsequent protection from immune-mediated mucosal diseasessuch as inflammatory bowel disease (IBD) and asthma. Invariant natural killer T (iNKT)cells recognize glycolipid antigens presented by the MHC class I-like protein CD1d andsecrete abundant amounts of proinflammatory cytokines such as IL-4 and IL-13 uponactivation We aimed to characterize the role of microbial exposure during early life and itseffects on tissue associated iNKT cells and their functions. Methods: Swiss Webster germ-free (GF) and specific pathogen free (SPF) mice assessed for the presence of iNKT cells byflow cytometry with CD1d tetramers at various time-points in multiple tissues. Chemokineligand and receptor expression was defined by qPCR, ELISA and/or immunohistochemistry.Colitis was assessed by oxazolone-induced colitis and asthma by ovalbumin (OVA)-inducedhypersensivity with or without anti-CD1d or anti-CXCL16 blockade. Results: Here we showthat, in GF mice, iNKT cells accumulate in the colonic lamina propria and lung tissue, butnot other tissues, resulting in a dramatic increase in morbidity in models of colitis andallergic asthma. Mechanistically, mucosal iNKT cell accumulation and iNKT cell-mediatedinflammation in GF mice was CD1d-dependent and associated with increased intestinal andpulmonary expression of the chemokine ligand CXCL16, leading to mucosal iNKT cellrecruitment, as it was blocked by anti-CXCL16 antibody treatment starting during neonatallife. Colonization of neonatal—but not adult—GF mice with a conventional microbiotaprotects animals from CXCL16-dependent, mucosal iNKT accumulation and subsequentsensitivity to CD1d-dependent and iNKT cell-mediated inflammation in the same modelsof colitis and allergic asthma. Conclusion: These results show that age-sensitive contact withcommensal microbes is critical in establishing mucosal tolerance to later environmentalexposures that stimulate iNKT cells and provide biologic support and a mechanistic basisfor the apparent protective effects of early-life associations with microbes.

Tu1963

Adrenergic Modulation of Inflammatory Dendritic CellsLaurens Nijhuis, Brenda Olivier, Francisca W. Hilbers, Wouter de Jonge

Introduction: Inappropriate antigen presenting cell (APC) reactivity can lead to pathogenicT cell polarisation and contribute to the pathogenesis of inflammatory bowel diseases (IBD).In the gut, APC such as resident dendritic cells (DC) are phenotypically and functionallyshaped by epithelial and stromal cell derived signals. However in addition, the sympatheticnervous system (SNS) is increasingly recognized as an additional regulatory factor in APCfunctions. The SNS innervates the gut mucosa and gut associated lymphoid tissue. Aimsand methods: The aim of this study was to asses if murine bone marrow derived DC (BMDC)are affected by sympathetic neurotransmitters and how adrenergic receptor expression isregulated in healthy and inflamed colons. Bone marrow was cultured for 7 days with GM-CSF to obtain immature DC (iDC). IDC were incubated for 15 minutes with epinephrineor with the beta2-adrenergic receptor (β2-AR) specific agonist salbutamol before 24 hoursof stimulation with LPS. IL-12p70, IL-6, TNFα and IL-10 production in supernatant wasmeasured by ELISA. Immunofluorescence stainings for the β2-AR and CD11c, CD11b asDC markers were performed. DC were analysed in colons from healthy, and chronic T celltransfer colitis in RAG1 -/- mice. Results: Short, 15 minutes incubation of BMDC withepinephrine or salbutamol (0.1 μM) prior to LPS stimulation of DC results in a decreasedproduction of IL-12p70 (6 pg/ml vs 110 pg/ml, p<0.0001), IL-6 (5500 pg/ml vs 12000 pg/ml, p<0.001) and TNFα (1100 pg/ml vs 450 pg/ml, p<0.001) compared to vehicle treatedDC. In contrast, IL-10 production increased 2 fold (320 pg/ml vs 150 pg/ml p<0.001). Thiseffect is completely blocked by the general β-AR antagonist propranolol or by the β2-ARspecific antagonist butoxamine. In control mice most CD11c+ cells are negative for β2-AR.However, in CD4+CD45RBhigh T cell-driven transfer colitis setting, we observed a significant

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