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sTu1837

The Notch Signaling Pathway Mediates Tight Junction Protein Stiochiometryin IBDDouglas R. Mathern, Lauren E. Laitman, Avantika Chitre, Lloyd Mayer, Stephanie Dahan

INTRODUCTION: Notch-1 regulates intestinal epithelial cell (IEC) growth and differenti-ation. We have previously shown that normal (NOR) lamina propria lymphocytes (LPLs)have the property of enhancing growth and differentiation and that Crohn's disease (CD)LPL have enhanced differentiating capacity. Cleaved Notch-1 is expressed in CD epitheliaextending from the surface to the crypt compared to NOR or ulcerative colitis (UC) mucosa.AIM: To elucidate the Notch ligand(s) responsible for the increase in Notch-1 activation inCD. METHODS: Intestinal resections from normal (NOR) (n=70), CD (n=33), or UC (n=22) patients were snap-frozen and subjected to Real-Time PCR for Notch-1, 2, DLL-1, 3,4, Jagged-1, 2, Occludin, Claudin-1, 3, 4, 5, 8. Human small bowel tissue sections wereimmunostained for Jagged-2. CD4+CD45RbHi naive T cells were isolated from C57BL/6(WT) spleens, adoptively transferred into RAG1-/- mice for 6 weeks and therefore they hadevidence of colitis. Colonic tissues were stained for Jagged-2 expression. RESULTS: Therewas a significant increase in Notch-1 but not Notch-2 mRNA in CD patients (p<0.05). Thisincrease was restricted to the small bowel. There was a significant increase in Jagged-2mRNA (but not in other Notch ligands) in the small bowel of CD patients (p<0.01).There was increased mRNA expression of occludin (p<0.01), claudin-1 (p<0.05), claudin-3 (p<0.001), claudin-5 (p<0.001), but not in claudin-4 and -8, in the small bowel of CDpatients, although claudin-8 mRNA was significantly increased in the right colon of CD andUC patients (respectively p<0.05). There was an increase in Jagged-2 staining in the laminapropria of small bowel tissue sections of CD mucosa. Moreover, upon transfer of CD45RbHiT cells into RAG deficient mice, expression of Jagged-2 was induced in the lamina propria.CONCLUSION: Increased Jagged-2 expression the RAG deficient mice transferred with naïveT cells speaks for a role of CD4 T cells in the maintenance of epithelial barrier integrity aswe have previously described. Increased Notch signaling pathway in CD potentially throughJagged-2 andNotch-1 could explain the enhanced epithelial differentiation that was associatedwith CD. Increased Notch-1 signaling pathway expression and activation could be responsiblefor abnormal tight junction stiochiometry in the mucosa of CD patients. Unbalanced tightjunction stiochiometry has been proposed to lead to a permeability defect. This couldpartially explain the loss of immune tolerance hypothesized in CD pathogenesis.

Tu1838

Inducible Heat Shock Protein 70 (HSP70) Maintains Intestinal HomeostasisDuring Acute and Chronic Colitis Through Modulation of Epithelial, Immuneand Microbial MechanismsYunwei Wang, Xiaorong Zhu, Sushila Dalal, Yun Tao, Mark W. Musch, Eugene B. Chang

Background: Inducible heat shock protein 70 (Hsp70) is a highly conserved, multifunctionalprotein. Our previous studies have shown that colonic Hsp70 expression is importantto maintain gastrointestinal homeostasis and down-regulation of Hsp70 in human andexperimental IBD renders the mucosa more susceptible to acute inflammation-associatedstress and cellular injury. However, the cell and molecular mechanisms by which Hsp70protects the intestine during inflammation-induced stress are not clear. In this study, severalpotential mechanisms of Hsp70-regulated intestinal mucosal protection were explored.Methods: Susceptibility to DSS stress-induced colitis was compared between wild type (WT)and Hsp70-/- mice. In addition, the role of Hsp70 in maintaining intestinal homeostasis inchronic colitis was examined in Hsp70/IL-10 double knockout (DKO) mice (all on C57Bl/6 background). Colonic epithelial barrier integrity was investigated by using permeabilityassay. Colonic tissue cytokine expression in mice was tested by ELISA. To investigate theeffect of Hsp70 on the gut microbiota which is essential in normal colonic development,enteric microbiota of Hsp70-/-mice was also examined by 16S rRNA gene based pyrosequenc-ing. Results: Hsp70-/- mice lost more body weight and had more severe colitis after 2.5%DSS treatment for 5 days compared to WT/heterozygous littermates (n=7). Gastrointestinalpermeability, as determined by serum FITC-dextran concentration 3 h after gavage, wasmarkedly increased in Hsp70-/- mice compared with controls (n=4) after DSS treatment.Cytokine secretion was also examined in colonic tissues. In Hsp70-/- mice two proinflammat-ory cytokines, IL-6 and TNF-α, were significantly increased. IL-10, in contrast, was dramatic-ally decreased in Hsp70-/- mice (p < 0.05) compared with that in controls. Role of Hsp70in gut homeostasis was also studied in Hsp70-/- /IL-10-/- mice. Our results showed thatDKO had an earlier onset (6 weeks old age) and higher percentage of colitis (100%)compared to IL10-/- mice (20%). As enteric microbiota play a role in development of colonicinflammation, the effects of Hsp70 on enteric microbiota were also examined in the study.Compared to litter mate controls, Hsp70-/- mice had significantly decreased colonic abund-ance of Tannerella, Alistipes, Oscillospira, Bifidobacterium and Allobaculum, but increasedButyricimonas and Ralstonia in their cecum. Conclusions: We demonstrate that Hsp70 playsa role in maintaining intestinal homeostasis by protecting colonic epithelial barrier integrity,maintaining immune balance, and affecting the assemblage of enteric microbiota. Theobserved decrease in Hsp70 expression in human IBD can potentially contribute to thepathogenesis, severity and chronicity of mucosal inflammation and immune activation.

Tu1839

Mapping Binding of CEACAM5 to CD8αGiulia Roda, Jianyu Xu, Clifford P. Stanners, Lloyd Mayer

Background: CD8+ regulatory T (TrE) cells can be activated by intestinal epithelial cells(IECs) through the complex formed by the non-classical I molecule, CD1d and gp180 bothexpressed on IECs. mAb B9, an anti-gp180 Ab, can block this induction. We previouslydemonstrated sequence homology between gp180 and CEACAM5 and that CEACAM5 hasproperties attributed to gp180, such as CD1d and CD8α binding Purpose of the study: Theaim of this study is to determine the B9 epitope on CEACAM5 and to map the binding ofCEACAM5 to CD8α. Methods: CHO cell lines were stably transfected with wild typeCEACAM5 and N domain mutants. An entire deletion of the N domain (ΔN), a deletion of

S-858AGA Abstracts

the N42 to I46 in the N domain (ΔNI), two conservative point mutations K35A and N42Dand a N70,81A mutation, which prevents N domain glycosylation, were selected. CHOtransfectants were stained with mAb B9. Wild type CEACAM5 and CEACAM5 N domainmutants soluble proteins were purified from supernatants obtained from PIPLC treatedtransfected CHO cell lines. CD8α soluble protein was purified from supernatants of trans-fected 293T cells. CEACAM5/CD8 binding affinity was measured by Biacore analysis. CD4recombinant protein was used as a negative control. A murine T cell line transfected withhuman CD8α, 3G8, was incubated with wild type CEACAM5 and the N70,81A mutantCEACAM5 peptides to assess their ability to activate CD8 associated p56lck. Results: TheΔN as well as the ΔNI deletion completely abrogated B9 reactivity. N70,81A >>> K35A =N42D mutant transfected CHO cells also showed a consistent decrease in B9 staining. Biacoreanalysis revealed that CEACAM5 binds to CD8α (not CD4) with an affinity comparable toMHC class I. The ΔN, ΔNI-CEACAM5, K35A and N42D mutants bind CD8α with a loweraffinity than the wild type CEACAM5. The loss of glycosylation of the N domain resultedin the greatest loss of affinity for CD8α. Furthermore, the N70,81A CEACAM5 appearedto lose the capacity to induce the phosphorylation of lck when compared to CEACAM5.Conclusion: These studies indicate that B9 binds to CEACAM5 through the N domain andthat CEACAM5 interacts with CD8α at sites that are overlapping (not exclusively) with B9epitopes. Moreover our data points to the glycosylation site in the N domain for both B9staining and CEACAM5 binding to CD8α.

Tu1840

Role of Necroptosis in Crohns Disease: Caspase-8 Regulates IntestinalEpithelial Necroptosis and Terminal IleitisClaudia Günther, Eva Martini, Nadine Wittkopf, Kerstin Amann, Benno Weigmann,Helmut Neumann, Maximilian J. Waldner, Stephen M. Hedrick, Stefan Tenzer, Markus F.Neurath, Christoph Becker

Epithelial cell death is a hallmark of intestinal inflammation and has been discussed as apathogenic mechanism driving Crohns disease in humans. However, the regulation of epithe-lial cell death and its role in intestinal homeostasis remains poorly understood. Usingconditional knockout mice we demonstrate a critical role for caspase-8 in regulating nec-roptosis of intestinal epithelial cells (IEC) and terminal ileitis, reminiscent of Crohns disease.Casp8ΔIEC mice showed spontaneous inflammatory lesions in the terminal ileum but notin the colon. However these mice showed a high susceptibility to experimental inducedcolitis with severe epithelial erosions. This results in an excessive infiltration of bacteriathrough this cell layer into the lamina propria and an increased expression of proinflammatorycytokines. Treatment of Casp8ΔIEC mice with antibiotics decrease the number of bacteriain the gut and therefore partially rescued the animals from the severe course of colitis,suggesting dysregulated anti-microbial immune functions of the intestinal epithelium. Thisconclusion was supported by the identification of an increasing amount of bacteria directlyattached to the intestinal epithelium in Casp8ΔIEC mice. This defect in antimicrobial hostdefence is mediated by a reduced expression of antimicrobial peptides produced by Panethcells. In line with this, Casp8ΔIEC mice lacked Paneth cells and instead showed an enormousamount of dying intestinal epithelial cells in this area. Histological analysis identified thisform of cell death as necroptosis, since dying cells showed typical features of necrosis,instead of apoptosis. Moreover epithelial cell death was associated with increased expressionof receptor-interacting protein 3 (RIP3), a key member of necrotic cell death in Casp8ΔIECmice and was inhibited upon blockade of necroptosis. Additionally we could detect RIP3staining colocalized with markers for Paneth cells. Our data suggest that deficient caspase8expression renders Paneth cells susceptible to necroptosis. Finally, we identified high levelsof RIP3 in human Paneth cells and increased necroptosis in the terminal ileum of patientswith Crohn's disease, suggesting a potential role of necroptosis in the pathogenesis of thisdisease. Additionally we were able to significantly reduce TNF-α-induced cell death of ilealbiopsies from control patients by pre-treatment with necrostatin-1 (nec-1), an inhibitor ofthe RIP1 kinase. Taken together, our data demonstrate a critical function of caspase8 inregulating intestinal homeostasis and in protecting IEC from TNF-α induced necroptoticcell death. As anti-TNF-α treatment is successfully used in the therapy of patients with Crohnsdisease, we hypothesized that one possible molecular mechanism of anti-TNF therapeutics isthe reduction of Paneth cell necroptosis in humans.

Tu1841

Epithelial Cell Signaling as Target for Antimicrobial Therapy AgainstDiarrheagenic Escherichia coli - New Role for Kinase Inhibitors in BlockingInfections With Enteropathogenic E. coliCarolin F. Manthey, Lars Eckmann

Diarrheagenic enteropathogenic Escherichia coli (EPEC) can contaminate food and watersupplies and cause serious diarrheal illness with high mortality. EPEC infections cause waterydiarrhea with potentially high mortality in infants, especially in developing countries. EPECand Citrobacter rodentium, a related mouse pathogen, are classified as attaching and effacingpathogens (A/E pathogens), as they can adhere to intestinal epithelial cells, destroy theirmicrovilli and induce characteristic actin-filled membranous pedestals. EPEC binding tointestinal epithelial cells induces a signaling cascade involving several host cell tyrosinekinases, which phosphorylate bacterial Tir (translocated intimin receptor) at position Y474and lead to recruitment of other host factors such as Nck, N-WASP and the Arp2/3 complex,and to actin polymerization and pedestal formation. Several host cell kinases are involvedin this process, including c-Fyn, Src, Yes and the ABL-family kinases ABL1 and ABL2 (ARG).ABL and ARG were shown to be sufficient, but not necessary, for pedestal formation in cellculture, suggesting that EPEC employs kinases in a redundant way to ensure phosphorylationof Tir. However, the physiological relevance of these cell culture observations has remainedunclear. Here we show that infection of mice with Citrobacter rodentium can be blocked for>7 days by treatment with the ABL kinase inhibitor, Imatinib mesylate, a drug used in thetreatment of CML patients. In exploring the basis for this observation, we found that Imatinibwas able to block attachment of EPEC to a model epithelium in cell culture under dynamicconditions (involving modest agitation, low bacterial inocula, and shortened infection times)designed to mimic transient bacteria-host interactions likely to occur in the intestinal tract.