In addition, TNFα protein release in the conditioned media was measured with an enzyme-linked immunosorbent assay (ELISA). KB-R7785(10μM) was used to inhibit ADAMs' activit-ies. RNA interference was performed to knock down endogenous ADAM 17 and AREG-regulating protein(ARP) 36. The association of ARP36 with ADAM17 was analyzed with IPwith anti-ARP36 antibody followed by western blotting(WB) with anti-ADAM17 antibody.Results: Themembrane protein immunoprecipitated with anti-proAREG antibody was identi-fied as ARP36 with MALDI/TOFMS analysis. ELISA and AP assay showed IL-1β-inducedTNFα shedding in HCT116 and HT29 cells and that TPA did TNFα shedding in U937cells within 5 hrs. KB-R7785 and depletion of ADAM17 significantly blocked TNFα sheddingwith IL-1β and TPA. Depletion of ARP36 significantly inhibited TNFα shedding with IL-1β and TPA in ELISA and AP assay. On the other hand knockdown of ARP36 did notabrogate the shedding of AREG and HB-EGF for which ADAM17 is responsible. IP and WBshowed the association of ARP36 with ADAM17. Conclusions: ADAM17 is closely associatedwith APR36. ADAM17 is involved in IL-1β-induced TNFα shedding in colon epithelial cellsand TPA-induced TNFα shedding in monocytes. ARP36 plays an important role on TNFshedding with IL-1β and TPA. Inhibition of ARP36 may be a new therapeutic strategy forprevention of TNFα shedding during IBD inflammation.

Mo1107

Progastrin Inhibits the Homotypic Fusion Between Human MacrophagesCarlos Hernandez, Dolores Ortiz-Masia, Dolores Barrachina, SARA CALATAYUD

Macrophages have the ability to undergo homotypic fusion and generate multinucleatedcells in chronic inflammatory processes with the participation of IL4. Macrophages can alsofuse heterotypically with cancer cells, which could gain the ability to migrate and inducemetastasis through this process (Cancer Metastasis Rev 2010, 29:695; Cancer Res 2009,69:8536). The molecular mechanisms implicated in both processes seem to be similar (NatRev Cancer 2008, 8:377). We studied the effects of progastrin on macrophages becausecolonic tumors synthesize progastrin and macrophages express annexin-II, a molecule thatserves as progastrin receptor. In the present study we report the serendipitous finding ofan inhibitory effect of progastrin on macrophage fusion. The effects of progastrin wereanalyzed on macrophages derived from human peripheral blood monocytes. Macrophageswere treated with progastrin (10-9-10-7M) or its vehicle in the presence of: 1) E.coli LPS(1μg/ml) plus IFNγ (20ng/ml), 24h, to induce pro-inflammatory macrophages (M1); 2) IL4(20ng/ml, 48h) to induce tolerogenic macrophages (M2a); or 3) IL10 (20ng/ml, 48 h) toinduce anti-inflammatory macrophages (M2c). Macrophages were incubated with hoeschtto identify the nuclei and with fluorescent labeled Ab against the surface molecules CD86(M1) and CD206 (M2a, M2c). Fluorescence was analyzed with the software for staticcytometry ScanR (>5000 cells/well). Hoescht fluorescence was used to record the spatialdisposition of cells, distinguishing between isolated and aggregated/multinucleated cells.The analysis of the fluorescence emitted by the surface markers allowed us to discriminatebetween independent cells in close proximity and multinucleated cells. All activation treat-ments induced a slight reduction in the number of cells per area (LPS+IFNγ: 93±3, IL4:90±2, IL10: 97±3% of control values) which was not modified by cotreatment with progastrin(10-7M; 90±4%, 90±2% and 87±7% respectively). The cytometric analysis detected thepresence of spontaneously generated multinucleated formations including 3-10 nuclei (mean:3.4±0.2). IL4 significantly increased the number of these formations (142± 6 %, p<0.05vs control values). IL10 did not induce any significant change (94±8%) while LPS+IFNγsignificantly reduced its presence (70±7%, p<0.05 vs control values). Co-treatment withprogastrin significantly reduced macrophage fusion in all cases (see figure). None of thesetreatments induced a significant effect in the mean size of these multinucleated forms.Our results indicate that progastrin inhibits macrophage fusion. This effect takes placeindependently of the pattern of macrophage activation. We hypothesized that, if this inhibit-ory effect of progastrin is also effective against macrophage fusion with cancer cells, thepeptide may constitute a neutralizing force against metastasis.

Effect of progastrin on macrophage fusion. * p<0.05 vs corresponding control group (RM-ANOVA + Newman-Keuls test).

Mo1108

Gastric Sonic Hedgehog Acts as a Macrophage Chemoattractant During theDevelopment of the Immune Response to Helicobacter pylori InfectionMichael A. Schumacher, Jessica M. Donnelly, Amy C. Engevik, Chang Xiao, Li Yang,Susan H. Kenny, Andrea Varro, Frederic Hollande, Linda C. Samuelson, Yana Zavros

Background: Macrophages are essential innate responders to Helicobacter pylori (H. pylori)-derived signals from the epithelium that are crucial to the development of gastritis. SonicHedgehog (Shh), a regulator of gastric epithelial differentiation and function, is known toregulate the host gastric immune response, but the precise nature of its role is unknown.Hypothesis: Gastric Shh acts as a macrophage chemoattractant during the innate immune

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response to H. pylori infection. Methods: Mice expressing a parietal cell-specific deletion ofShh (PC-ShhKO) and controls were infected with H. pylori. Gastric Shh, cytokine andchemokine expression was assayed by qRT-PCR or by a Luminex®-based multiplex assay2, 7 and 180 days post-inoculation. Circulating Shh concentrations were assayed by ELISA.To identify the direct role of Shh signaling in macrophage recruitment to the stomach, weperformed bonemarrow chimera experiments usingmice with a myeloid cell-specific deletionof Hedgehog signal transduction protein smoothened (LyzsCre/SmoKO). Macrophage recruit-ment in response to H. pylori was measured in both gastric tissue and peripheral blood byfluorescence-activated cell sorting (FACS). Results: 1) H. pylori-infected PC-ShhKO mice failto develop gastritis: Control mice infected with H. pylori for 6 months developed a significantinflammatory response that was documented by CD4+ T-lymphocyte infiltration and eleva-tion of pro-inflammatory cytokines IFNγ and IL-1β in the stomach. PC-ShhKO mice did notdevelop gastric inflammation even after 6 months of H. pylori infection. In control stomachsShh expression was induced within 7 days post-infection. 2) Gastric Shh acts as a macrophagechemoattractant in response toH. pylori infection: Control mice exhibited elevated concentra-tions of the neutrophil chemoattractant KC, a murine IL-8 homologue, monocyte chemotacticprotein-1 (MCP-1) and circulating Shh concentrations that were accompanied by the recruit-ment of CD11b+F4/80+Ly6Chigh macrophages 48 hours post-infection. Control recipientmice transplanted with bone marrow from control animals exhibited an influx of CD11b+F4/80+Ly6Chigh macrophages to the gastric mucosa that was accompanied by a significantdecrease in the number of macrophages in the periphery. H. pylori-infected control recipientmice transplanted with LyzsCre/SmoKO bone marrow donor cells showed an absence ofCD11b+F4/80+Ly6Chigh macrophage recruitment. Conclusion: During the innate immuneresponseH. pylori-induced Shh released from the stomach acts as a macrophage chemoattract-ant to play a role in the initiation of gastritis. Loss of Shh, as observed during gastritis, mayresult in the disruption of a sufficient immune mechanism necessary to eradicate bacterialinfection, and thus allowing the development of H. pylori-induced chronic inflammation.

Mo1109

Immune Regulation of the Intestinal Progenitor Cell MicroenvironmentTim Vanuytsel, Luigi Notari, Shu Yan, Aiping Zhao, Jennifer A. Bohl, Leon McLean, RexSun, Joseph F. Urban, Allen Smith, Jan F. Tack, Terez Shea-Donohue

Background: The gastrointestinal tract is the largest interface between the external andinternal milieux. To adapt to different environmental challenges, the epithelium renewsevery 5 to 7 days. Enteric infections induce a proliferative response in the colon; however,knowledge of how different cytokine profiles in the stem-cell microenvironment influenceprogenitor cell proliferation and migration is limited. Aim: To elucidate the effect of differentcytokine microenvironments on intestinal progenitor cells.Materials and methods: C57BL/6 mice (N=25) were infected with Citrobacter rodentium (Cr), a model for enterotoxigenicE. coli, by oral gavage of 1010 CFU and studied every week (n=5/group) for 4 weeks postinfection (PI). BALB/c and STAT6 knockout (KO) (N=20/group) were infected with thececal-dwelling Trichurismuris, amodel for humanwhipworm, by gavage of 200 embryonatedeggs and studied every week (n=5/group) for 4 weeks. All mice were injected with 10mgbromodeoxyuridine (BrdU) 2 hours before sacrifice. Colonic segments were processed forhistology and gene expression. BrdU positive cells per crypt were counted and the mucosalheight measured in >10 well-oriented crypts/mouse. Gene expression of IFNγ (Th1), IL17A(Th17), IL4 and IL13 (Th2) and CXCL10 was quantified by real-time PCR. Results: Crupregulated IFNγ and IL17A, cytokines needed for Cr clearance by day 14 PI (Table 1)coinciding with a proliferative response of the colonic epithelium indicated by an increasein the number of BrdU positive cells per crypt (13.9±1.6 vs. 5.2±0.7; p<0.01) and in mucosalheight (191±6 vs. 119±6 μm; p<0.01). In WT T. muris infected mice , the maximal increaseof IL4 and IL13, cytokines needed for clearance, occured at day 14 PI and coincided witha modest upregulation of IFNγ and CXCL10 (Table 1). At this time there was also a transientincrease in BrdU positive cells per crypt (11±0.6 vs. 4.9±0.3; p<0.01) and mucosal height(131±10 vs. 97±2 μm; p<0.01) preceding clearance. In contrast, day 14 PI STAT6 KO micelacked upregulation of Th2 or Th1 cytokines or increased epithelial proliferation despitethe presence of worms. These mice develop a chronic infection with a proliferative responseat day 28 PI that also coincides with the generation of a Th1 predominant response (Table1) and CXCL10 upregulation. Conclusion: By manipulating the colon cytokine micro-environment with two polarized infections in different backgrounds, we showed that themaximal proliferative response coincides with the mounting of a Th1 cytokine response andproduction of CXCL10. The latter is upregulated in IBD-patients and is a well-knowninhibitor of epithelial cell migration which leads to crypt hyperplasia in the presence ofproliferation (Cliffe et al. 2005). Our findings provide further insight in the immune-regulation of the progenitor cell microenvironment of the colon.

Changes in gene-expression of cytokines during enteric infections. Data expressed as averagefold-change±SEM. #p<0.05; *p<0.01

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