TLR2 stimulation, and concordantly, depletion of optineurin in THP-1 cells was associatedwith reduced TNF release after TLR2 activation. Conclusions: In conclusion, we havedeveloped a successful strategy to identify abnormal expression of genes in individual CDpatients relevant to diminished macrophage function. In a wider context, this approach maybe equally applicable in delineating the molecular pathogenesis of other complex disorders.1. A. Franke et al., Nat. Genet. 42, 1118 (2010) 2. A. M. Smith et al., J. Exp. Med. 206,1883 (2009)

Tu1968

Increased Sensitivity to TGFβ Results in Defects in T Cell Proliferation andRegulatory T Cell Induction in the Absence of Wiskott-Aldrich SyndromeProtein (WASp)Elisa K. Boden, David Dunkin, Jeremy A. Goettel, Osub Ahmed, Christopher J. Moran,Deanna D. Nguyen, Lloyd Mayer, Scott B. Snapper

Background: The Wiskott-Aldrich Syndrome is an immunodeficiency that results frommutations in WASp. WASp transduces cell surface receptor signals to the actin cytoskeletonvia interaction with ARP2/3, a known risk allele for ulcerative colitis. Both humans andmice withWASp-deficiencymay develop colitis associated with reduced number and functionof CD4+CD25+Foxp3+ regulatory T cells (Tregs). Here we demonstrate altered de novoinduction of Tregs and reduced T cell proliferation in the absence of WASp that correlateswith increased responsiveness to TGFβ. Methods: Naïve T cells from ova-specific TCR-transgenic wild type (WT) or WASp-deficient(WKO) mice were cultured with ova peptideand TGFβ and analyzed for FoxP3 and CFSE dilution at 72h. WT or WKO T cells wereexposed to TGFβ for 0-60 minutes and lysates were fractionated by SDS-PAGE. Westernblotting was performed for phospho-SMAD2, total SMAD2, CD3ε and TGFβrI/II. Non-transgenic WT and WKO mice were fed ova daily for 5 days followed by immunizationand boosting with ova. Delayed-type hypersensitivity (DTH) was assessed by injection ofova into the L footpad and PBS into the right footpad. At 24h DTH was calculated as thedifference in size between the left and right footpad. Draining LNs were restimulated withova and IFNγ from the culture supernatants was measured by ELISA at 48h. Results:Stimulation of naïve WKO T cells resulted in reduced proliferative response to antigencompared with WT. This correlated with an altered threshold for the maximal induction ofTregs, such that the peak induction of Tregs in WKO T cells required 10-fold increasedantigen dose. When responsiveness to TGFβ was assessed, WKO T cells demonstratedincreased phosphorylation of the TGFβ receptor signaling protein, SMAD2, at early timepo-ints compared to WT. Expression of TGFβ receptor was not altered in WKO T cells.Surprisingly, oral tolerance to low-dose antigen remained intact in WKO mice. WKO micefed oral ovalbumin had no DTH response to intradermal ovalbumin. Additionally, followingimmunization, the draining lymph nodes of tolerized mice had complete suppression ofIFNγ response to restimulation with ovalbumin. Conclusions: These data implicate increasedsensitivity to TGFβ as a novel mechanism for the reduced Tregs and development of colitisin the absence of WASp. In the setting of oral tolerance, a process known to depend oninduction of Tregs and on direct effects of TGFβ, there is no defect in WKO animals. Wehypothesize that defects in Treg induction may be compensated by increased sensitivity tothe direct effects of TGFβ. Further understanding of the mechanisms that govern Treghomeostasis may allow for the development of Treg-targeted therapies for IBD.

Tu1969

Elevated Expression of the Cytosolic DNA Sensors AIM2 and ZBP1/DAI inActive Ulcerative Colitis but Not Crohn's Disease Colonic TissueAine Fanning, Gerard Moloney, John MacSharry, Monica Ambrose, Eamonn M. Quigley,Fergus Shanahan, Silvia Melgar, Kenneth Nally

Background: Ulcerative colitis (UC) and Crohn's disease (CD) are chronic remitting andrelapsing inflammatory diseases of the gastrointestinal tract. While both are distinct andseparate inflammatory conditions, their pathophysiology is still not well defined. For example,while the aetiology of both UC and CD is largely unknown, it is suggested that the interplaybetween host genetic susceptibility, the external environment, the intestinal microflora anda deregulated immune response in combination, contribute to general disease pathogenesisfor both conditions. Pattern recognition receptors (PRRs) are used by the innate immunesystem to detect common Microbe-Associated Molecular Patterns (MAMPs) and endogenousDanger-Associated Molecular Patterns (DAMPs). Hence, different PRRs can distinguish differ-ent MAMPs and DAMPs thereby directing the selectivity of the subsequent immune responseto specific exogenous and endogenous triggers. Aims: We profiled the expression of 50known PRRs including C-type lectin receptors (CLRs), Toll-like Receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs) and AIM2-like receptors (ALRs) in healthyand IBD tissue to gain further insight into the distinct pathogenesis of UC and CD. Methods:Total RNA was isolated from healthy (n=33), UC active (n=38), UC inactive (n=30), CDactive (n=8) and CD inactive (n=10) colonic mucosal biopsy tissue. The expression of apanel of 50 known PRRs were screened by qRT-PCR in a small panel (n=8-12 per diseasegroup) of individual patient samples and differentially expressed genes were subsequentlyvalidated in cDNA from remaining tissue samples. Because activation of cytosolic DNAsensor pathways typically leads to a Type I Interferon (IFN) response we screened forexpression of a panel of IFN responsive genes by qRT-PCR in the same set of cDNAs.Results: From the initial screen, it was notable that there was a selective up-regulation ofthe cytosolic DNA sensor genes AIM2 and ZBP-1/DAI in active UC but not inactive UC,active CD or healthy tissue. IFI16 was also strongly up-regulated in active and inactive UCbut to a lesser degree than AIM2 and ZBP1/DAI. The differential expression of these geneswas confirmed in a larger number of patient samples. In addition, we found several type IIFN responsive genes to be up-regulated in active UC. Conclusion: The expression of thecytosolic DNA sensors - AIM2, ZBP-1/DAI, IFI16 - are selectively and strongly up-regulatedin active UC but not active CD or healthy colonic tissue samples. In addition analysis ofType I IFN regulated genes suggests that these DNA sensing pathways are active in UC.Therefore, activation of cytosolic DNA sensors by DNA from viral, bacterial or host genomescould distinguish UC from CD pathogenesis.

S-889 AGA Abstracts

Tu1970

Givinostat Induces an M2 Macrophage Phenotype and Modulates IL-6SignalingMartin Wetzel, Marco Gerling, Rainer Glauben, Thorsten Stroh, Ulrike Erben, MartinZeitz, Britta Siegmund

Background: The histone deacetylase (HDAC) pan-inhibitor givinostat (ITF2357) currentlyundergoes clinical phase II trials in juvenile idiopathic arthritis patients. In murine modelsof colitis, givinostat ameliorates disease activity and protects from colitis-associated carcino-genesis. Recently, the HDAC inhibitors trichostatin A and valproic acid were shown tomarkedly alter macrophage phenotypes. Here, we studied whether altered macrophagephenotypes and functions might contribute to the diverse clinical effects of givinostat.Methods and Results: Bone marrow-derived macrophages (BMDM) obtained from femursof C57BL/6 mice were cultured in the presence of macrophage-colony stimulating factor.Mature F4/80+ CD11b+ BMDM were treated with 0 to 200 nM givinostat, followed bystimulation with lipopolysaccharide (LPS). As measured using antibody-based methods,secretion of tumor necrosis factor, IL-6, IL-10, IL-12p70, and the soluble IL-6 receptor(sIL6R) was reduced by givinostat in a dose-dependent manner; contrarily, monocyte chemot-actic protein-1 (murine analogue of CCL2) was increased. Cytometric analyses showed thatgivinostat stifled the LPS-driven up-regulation of IL-6R alpha chain (CD126), while leavingits signal transducer CD130 unchanged. Givinostat reduced the capacity of BMDM forantigen-specific activation of CD4+ T cells without affecting antigen processing, as shownby T cell proliferation and interferon gamma secretion after co-culture of BMDM with naïveCD4+ T cells from OT-II mice in the presence of ovalbumin protein or OVA323-329peptide. Phagocytosis assay using fluorescent latex spheres revealed significantly impairedphagocytotic activity of givinostat-treated BMDM. Conclusion: We here demonstrate thatgivinostat treatment alters the phenotype of BMDM, leading to an impairment of majorhistocompatibility complex class II-dependent T cell activation and of phagocytotic activity.IL-6 signaling is affected at the levels of cytokine secretion, receptor expression and bydiminishing IL-6 trans-signalling via sIL 6R. Taken together, our results suggest the inductionof an M2-like phenotype by therapeutic levels of givinostat. These effects on central macroph-age functions by HDAC pan-inhibition warrant further investigation In Vivo.

Tu1971

Anti-TNF Induced Regulatory Macrophages Display High Levels of AutophagyAnne Christine Vos, Manon Wildenberg, Alon D. Levin, Gijs R. van den Brink, Daniel W.Hommes

Background One of the most exciting developments in IBD therapy has been the introductionof anti-TNF agents. Although originally believed to function through neutralization of solubleTNF, we have previously shown that anti-TNF agents induce regulatory macrophages whichhave immune-suppressive and wound healing capacity (Vos et al, Gastroenterology 2010).How these cells exert their effects has not been established thus far. Over the past few years,genome-wide association studies have shown a correlation between Crohn's Disease (CD)and a number of autophagy-related genes, implicating a role for autophagy dysfunction in thepathogenesis. We evaluated the induction of autophagy in anti-TNF induced macrophages.Methods Mixed lymphocyte reactions were established using PBMC from two healthy donorsin a 1:1 ratio and treated with anti-TNF or IgG control. Subsequently, macrophages wereisolated from the cultures using CD14-microbeads. Levels of autophagy were determinedby enumeration of LC3+ spots and quantification of LC3-I/LC3-II ratio's. In addition, expres-sion levels of various autophagy-related genes were studied by gene array. Results Anti-TNF induced regulatory macrophages displayed increased numbers of autophagosomes asindicated by LC3+ spots. In addition, conversion of the autophagosomal protein LC3 wasincreased in anti-TNF treated cultures compared to IgG treated cultures. Finally, expressionof a number of autophagy related genes including atg5, atg7, atg9 and atg16l2 were upregul-ated in the induced regulatory macrophages in comparison to inflammatory M1 type macro-phages. Conclusion Regulatory macrophages induced by anti-TNF therapy display increasedlevels of autophagy compared to IgG-induced or inflammatory macrophages. Given the rolefor impaired autophagy in the pathogenesis of CD, this may be an addition mechanism bywhich anti-TNF compounds exert their beneficial effects.

Tu1972

Mice Overexpressing Wild-Type Human Alpha-Synuclein Display Alterationsin Colonic Myenteric Ganglia and Propulsive Colonic Motor FunctionLixin Wang, Iddo Magen, Franziska Richter, Pu-Qing Yuan, Marie-Francoise Chesselet,Yvette Tache

Background: Gastrointestinal (GI) symptoms are one of the most prevalent non-motorsymptoms of Parkinson's disease (PD), and significantly impact patients' quality of life.Despite some recent progress, there remains a paucity of suitable animal models to studythe mechanisms of PD-related GI symptoms and test new treatments. Alpha-synulein, aprotein associated with both familial and sporadic PD forms pathological neuronal aggregatesin PD, including in myenteric neurons. Therefore, mice overexpressing α-synuclein mayprovide a suitable model for assessing PD-related GI deficits. We have previously showncolonic dysmotility in 12 months old mice overexpressing human wild type α-synucleinunder the Thy1 promoter (Thy1-aSyn). Aim: To investigate gut alterations in Thy1-aSynmice at earlier ages as well as expression of α-synuclein and its relationship to otherneurotransmitters in the distal colon. Methods: Defecation, gastric emptying (GE) and immu-noreactivity of α-synuclein, peripheral choline acetyl transferase (pChAT), tyrosinehydroxylase (TH), neuronal nitric oxide synthase (nNOS) and vasoactive intestinal peptide(VIP) in distal colon myenteric plexi were assessed in Thy1-aSyn mice. Results: Thy1-aSynmice aged 2.5-3 or 7-8 months old had 81% and 55% reduction in fecal pellet output,respectively in response to the first 15 min of 2 h in novelty, and 60% and 69% reduction,respectively during the subsequent 1-h refeeding compared with age matchedWT littermates.The reduction of fecal pellets was not correlated to food intake during the 1 h of refeeding(r2 = 0.03, p > 0.05). In the early dark phase 8 months old Thy1-aSyn mice showed a 59%

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