blood monocytes to evaluate the effects of these mAbs (and a control mAb) against toxin A-and B-associated proinflammatory responses and epithelial cell damage. Results: Incubation ofPBMCs for 16 hr with toxin A (0.1 μg/ml) or toxin B (1 μg/ml) increased TNF α (p=0.01 &p=0.006) and IL-1β (p=0.0001 & p=0.006) protein in conditioned media, as well as TNF α(p=0.0001 & p=0.0004) and IL-1β (p=0.0001 for both toxins) mRNA expression. Incubationof anti-toxin A (MK3415) or anti-toxin B (MK6072) mAbs (30 μg/ml) with human PBMCsinhibited toxin A- and toxin B- mediated TNF α (by 63%, p=0.006, MK3415 & by 66%,p=0.0239, MK6072) and IL-1β (by 90%, p=0.0001, MK3415& by 40%, p=0.0005, MK6072)protein release within 4 hours. MK3415 and MK6072 also significantly diminished toxinA- and toxin B- mediated NF-κB p65 phosphorylation (by 40%, p=0.008, MK3415 & by30%, p=0.0034, MK6072) in human monocytes, respectively within 1 hour. Incubation ofhuman colonic tissues with toxin A and toxin B resulted in significant increase of TNF α(p=0.0038 & p=0.0019, respectively) and IL-1β (p=0.0001 & p=0.04, respectively) mRNAexpression and increased epithelial cell damage (2 fold increase, p ,0.01 by both toxins)in 16 hours. MK3415 and MK6072 significantly reduced toxin A- (1 μg/ml) and B- (10 μg/ml) induced TNFα (by 50%, p=0.01, MK3415; by 55%, p=0.003, MK6072) and IL-1 β (by90%, p=0.0001, MK3415 and by 95%, p=0.0001, MK6072) mRNA expression as well asepithelial cell damage in human colonic tissues (by 50%, p=0.0001, both antibodies againstrespective toxins) in 16 hours. Conclusions: Our results underscore the effectiveness ofMK3415 and MK6072 in preventing C. difficile toxin A- and toxin B-mediated inflammatoryresponses and epithelial cell damage in human colon providing a possible mechanism fortheir effectiveness in recurrent CDI. Grant support: Merck Pharmaceuticals (CP); Crohn'sand Colitis Foundation of America Career Development Award (HWK) and Student ResearchFellowship (SH); NIH K01 DK084256 (HWK), and DK046763 (DQS and SRT).

137

Intra-Luminal Interleukin (IL)-27 Ameliorates Acute Murine TNBS Colitis - APotential Future Human Therapeutic for Inflammatory Bowel Disease?Mairi H. McLean, Miranda L. Hanson, Lothar Steidler, Miriam R. Anver, Wei Shen, ScottK. Durum

IL-27 is a heterodimeric cytokine that exerts anti-inflammatory properties. Lactococcus lactis(L. lactis), a bacterium used in the food industry has been engineered to deliver IL-27 intra-luminally to the gut following oral ingestion. This study assessed the therapeutic efficacyof L.lactis-IL-27 (LL-IL-27) in acute 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis. 2mgTNBS in 45% ethanol or 45% ethanol alone was delivered intra-rectally to 6-8 week oldmale SJL mice. L. lactis control or LL-IL-27 was delivered by oral gavage on 4 occasions,24 hours apart, commencing on the day of colitis induction. Tissues were harvested 24hours after the final gavage, 4 days after TNBS instillation, or at time of death. Blood wascollected 2 days post TNBS instillation. Clinical data was collected daily, macroscopic andmicroscopic scores of colitis were obtained, and RNA and protein was extracted from distalcolon homogenate. Inflammatory (Il6, Il1b, Tnf, Ifng, Il10, Il17a, Foxp3, Il13, Il12) andepithelial barrier integrity (occludin, claudin 2 & 4, Muc1, Muc2, cadherin, trefoil factor3, tight junctional protein 1) gene expression was assessed by RT-PCR. Inflammatory proteinconcentration (IL-6, IL-10, IL-1β, IFN-γ, TNF) was determined by ELISA. Distal colonmacrophage and neutrophil infiltrate was assessed by immunohistochemical staining for F4/80 and myeloperoxidase, respectively. TNBS resulted in a rapid acute severe distal colitis.Treatment with LL-IL-27 led to a significant reduction in disease activity index comparedto L. lactis control (4.9 vs. 8.7/12 on day 2 (p=0.001) and 3.6 vs. 7.7/12 on day 3 (p=0.001), respectively), decreased colonic weight (p=0.02), increased colonic length (p,0.001),reduction in serum CRP (p=0.003) and survival advantage (p ,0.001, day 4). There was amodest reduction in histological colitis score (10.5 vs. 8/12, p= 0.017). Severity of inflamma-tory cell infiltrate remained static. However, there was a significant improvement in mucosalulceration (2.3/4 vs. 4/4, p=0.008). Despite this, there was no dramatic differential expressionof genes involved in epithelial barrier integrity. There was a significant increase in expressionof IL-6, IL1-β, TNF and IFN-γ in TNBS treated mice with no differential effect seen withLL-IL-27. However, LL-IL-27 led to a significant reduction in IL-6 (p=0.002), IL-1 β (p=0.001) and TNF (p=0.014) protein, suggesting a potential influence on post-translationalmodification. Macrophage infiltrate increased with TNBS treatment, with no significantchange with LL-IL-27. There was however a significant reduction in neutrophil infiltrate inthe LL-IL-27 group compared to L. Lactis control (p=0.004). In conclusion, intra-luminalIL-27 ameliorates acute murine TNBS colitis and may be a future potential therapy forchronic inflammatory bowel disease and acute colitis of differing etiologies in humans.

138

Use of CEACAM5 Synthetic Peptide Combinatorial Library to CharacterizeBinding of CEACAM5 to CD8αGiulia Roda, 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 have previouslydemonstrated sequence homology between gp180 and CEACAM5. Furthermore, CEACAM5has properties attributed to gp180, such as CD1d and CD8 α binding. We suggest thatunique set of interactions between CEACAM5, CD1d and CD8 α can render CD1d moreclass I-like, facilitating antigen presentation by CD1d to T cells and the activation of CD8+Tregs. Purpose of the study: The aim of this study is to determine where the binding ofCEACAM5 to CD8 occurs and which residues are functional for the activation of CD8+Tregs. Methods: CHO cell lines were stably transfected with CEACAM5 wild type and Ndomain mutants. An entire deletion of the N domain ( ΔN), a deletion of the N42 to I46(ΔNI), two conservative point mutations K35A and N42D, and a N70,81A mutation whichprevents N domain glycosylation were selected. Soluble proteins for wild type CEACAM5and CEACAM5 mutants were purified from supernatants obtained from PIPLC treatedtransfected cells. CD8α soluble protein was purified from supernatants of transfected 293Tcells. Biacore analysis was performed to evaluate CEACAM5/CD8 binding affinities. Anoverlapping peptide library of CEACAM5 N domain was generated to identify the aminoacids that contribute to its functionality (19 peptides with offset of 5). We tested the ability

S-33 AGA Abstracts

of the entire pool of peptides and of 3 combinations of peptides to phosphorylate CD8associated p56Lck kinase both in human isolated CD8+ T cells and in a murine T cell linetransfected with human CD8α, 3G8, using In-cell Western Blot assay. OKT8, an anti-CD8antibody, was used as positive control. Results: Biacore analysis revealed that CEACAM5binds to CD8α (not CD4) with an affinity comparable to MHC class I. The ΔN, ΔNI-CEACAM5, K35A and N42D mutants bind CD8α with a lower affinity than the wild typeCEACAM5. The loss of glycosylation of the N domain resulted in the greatest loss of affinityfor CD8α. B9, when premixed with full length CEACAM5, blocks CEACAM5/CD8 binding.Furthermore, the N70,81A CEACAM5 appeared to lose the capacity to induce the phosphory-lation of Lck when compared to CEACAM5. The entire collection of peptides inducedphosphorylation of Lck compared to OKT8. When pooled peptides were tested, pool 3 ..2(pool 2: G30-T55; pool 3: P66-Y107) resulted in the activation of CD8+ T cells. Conclusion:Our data suggest the crucial role of the N domain sugar bridge site in CEACAM5 bindingto CD8α and in the activation of CD8+ Tregs. Furthermore, we identified the regions ofthe N domain where the functional amino acid residues are located.

148

Human Intestinal Epithelium Derived From Induced Pluripotent Stem CellsAmanda L. Kauffman, Alexandra V. Gyurdieva, John R. Mabus, Pamela J. Hornby

BACKGROUND: Current understanding of human intestinal physiology and pathophysiologyrelies heavily upon cell-based in vitro models of intestinal epithelia. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer simplicity of culturing differentiatedintestinal cell types but do not recapitulate the endogenous expression of receptors andtransporters of the primary epithelial tissue. Isolated human intestinal epithelial cells retainthese features, but are difficult to culture and have limited viability. The advent of inducedpluripotent stem cells (iPSCs), adult somatic cells reprogrammed to an embryonic stem cell-like state, has unleashed a huge therapeutic potential, as signified by the 2012 Nobel Prizein Medicine awarded for this work. Taking advantage of this technology, we generated iPSC-derived human intestinal cells, analyzed expression of epithelial markers and performed apreliminary assessment of their functional utility. METHODS: Intestinal organogenesis meth-ods (Spence et al., 2011; Rezania, et al. 2012) were optimized to differentiate five retrovirallyand non-virally reprogrammed human iPSCs lines in 3D organoid or transwell cultures.Intestinal markers were evaluated by immunohistochemistry, flow cytometry and mRNAexpression. Monolayer integrity was assessed by Trans Epithelial Electrical Resistance (TEER)and FITC-Dextran permeability, and compared to Caco-2 and commercially-available humanprimary intestinal epithelial cells. RESULTS: All iPSC-derived cell lines resulted in intestinalorganoids with subsets of cells colocalizing E-cadherin (tight junctions), CDX2 (hindgutepithelia), and villin (enterocytes). However, frequency and intensity of co-localized markerexpression was greatest for two retrovirally reprogrammed cell lines. One of these cell lineswas successfully adapted to culture in transwells and demonstrated monolayer formationwith TEER values similar to primary cells (.1000 ohms/cm2) and better than Caco-2 cells(,250 ohms /cm2). Monoclonal antibodies engineered for different neonatal Fc Receptor(FcRn)-binding affinities were used to demonstrate FcRn-dependent binding to iPSC-derivedintestinal cells, which was comparable to human primary intestinal epithelial cells (~2.5-fold greater binding detected for high FcRn-binding variant relative to low-binding variant).CONCLUSIONS: iPSCs can be differentiated into intestinal-like cells that express matureepithelial markers, and are capable of forming tight monolayers in transwell cultures. Thus,iPSC-derived intestinal-like cells provide a novel tool for understanding normal humanepithelial physiology. This methodology could also be used to generate epithelial cells fromiPSCs derived from patients with intestinal disease to elucidate pathophysiological processes.

149

NFκB Mediates Helicobacter pylori-Induced Sonic Hedgehog Expression in theStomach: Use of a Novel In Vitro Model to Study Epithelial Response toInfectionMichael A. Schumacher, Eitaro Aihara, Rui Feng, Amy C. Engevik, Marshall H. Montrose,Yana Zavros

BACKGROUND: Helicobacter pylori (H. pylori) infection leads to increased expression andsecretion of Sonic hedgehog (Shh) in the stomach that is associated with the developmentof the host inflammatory response. The mechanism by which H. pylori induces Shh is notwell understood, but Shh is reported to be a target gene of transcription factor nuclearfactor-κB (NFκB). During H. pylori-induced gastritis, NFκB activation is critical for pro-inflammatory cytokine production. HYPOTHESIS: Induction of Shh by H. pylori infectionis mediated by NFκB activation. METHODS: To visualize Shh ligand expression in responseto H. pylori infection in vivo, we used a mouse model that expresses Shh fused to greenfluorescent protein (Shh::GFP mice) in place of wild-type Shh. GFP is inserted into the Shhprotein such that secreted ligand retains both GFP and lipid modifications required forsignaling. Changes in Shh expression were measured in 3-dimensonal structures of primarycultured mouse epithelial cells known as gastric organoids. Organoids were pre-treated forone hour with NFκB inhibitor or vehicle prior to microinjection with H. pylori. At threehours post-injection, Shh gene expression was analyzed by qRT-PCR. RESULTS: 1) H. pyloriinduces Shh ligand expression in vivo: Compared to uninfected controls, H. pylori infectioninduced expression of Shh in Shh::GFP mice within 2 days post-infection. Immunofluores-cence using cell lineage markers demonstrated that Shh ligand predominantly localized tothe parietal and surface pit mucous cells. 2) H. pylori-induced Shh expression is mediatedby NFκB signaling in vitro: In vivo studies investigating the interaction between H. pyloriand the gastric epithelium are limited in that the host response generates recruitment ofcells that may confound interpretation. Previous in vitro cell culture studies are limited inthat they do not replicate the complement of cells that make up the stomach. Thus, weused gastric organoid cultures as a gastric model to determine the involvement of theNFκB pathway in regulating Shh expression induced by H. pylori, but independent of therecruitment of circulating host cells. Expression of markers of all major gastric cell lineagesincluding the parietal cell marker HK-ATPase was observed in gastric organoid culturesdemonstrating a capacity for this model to be useful for these mechanistic studies. Microinjec-tion of gastric organoids with H. pylori resulted in a 3-fold induction in Shh expression that

AG

AA

bst

ract

s