Enterotoxigenic E. Coli (ETEC) Pathogenesis Modeled in Human Enteroid Monolayers Demonstrates MRP-Related Cyclic Nucleotide Secretion

197 ENTEROTOXIGENIC E. COLI (ETEC) PATHOGENESIS MODELED IN HUMAN ENTEROID MONOLAYERS DEMONSTRATES MRP-RELATED CYCLIC NUCLEOTIDE SECRETION Huimin Yu, Laxmi Sunuwar, James M. Fleckenstein, Mark Donowitz, Jennifer D. FoulkeAbel Background: The Global Enteric Multicenter Study (GEMS) determined that enterotoxigenic E. coli (ETEC) producing heat stable enterotoxin A (ST), with or without co-expression of heat labile enterotoxin (LT), is one of four leading pathogens worldwide that trigger acute diarrhea and associated mortalities in developing countries. ETEC is also a common cause of traveler's diarrhea. Mechanisms governing the pathophysiological response to ETEC infection in humans remain incompletely characterized, especially regarding STa-elicited fluid secretion via cGMP production. We employed human enteroid monolayers to evaluate how synthetic STa, ETEC strain H10407, and related ETEC mutants affect cGMP production in human jejunal epithelium. Methods: Human intestinal biopsies or surgical specimens were obtained through protocols approved by the Johns Hopkins University School of Medicine Institutional Review Board. Crypt isolation and 3-D culture methods were reported previously by our group (Gastroenterology 2016;150(3):638-649.e8). Human enteroid 2-D monolayers derived from 3-D enteroid culture were grown on collagen IV-coated Transwells and differentiated by removal of Wnt3a and R-spondin1 for 5 days. Confluency and differentiation were monitored by transepithelial electrical resistance measurements. Apical, basolateral, and intracellular cGMP was measured by ELISA. Results: To establish a functional ST-enteroid monolayer model, ETEC strains (109 cfu/mL) or synthetic STa preparations were incubated on the apical side of monolayers for 6 h followed by assay for cGMP content in the apical medium, basolateral medium, and intracellular fraction. cGMP was elevated in all three compartments, with the greatest accumulation occuring in the basolateral medium. The multidrug resistance protein (MRP) inhibitors MK571 (50 µM) or probenecid (1 mM) decreased secretion and increased intracellular sequestration of cGMP. ETEC deficient in LT expression induced less cGMP than the wild type strain in each of the three compartments, suggesting that ST production or activity is dependent upon the presence of LT. Conclusions: 1) In the human enteroid monolayer model, ST released by ETEC H10407 induces cGMP production that is comparable to a local luminal concentration within the range of 100 pM1 nM ST. 2) Increased intracellular cGMP is accompanied by apical and basolateral secretion of the molecule, with basolateral secretion being predominant. 3) cGMP secretion can be blocked by the MRP inhibitors MK571 or probenecid. 4) LT expression increased STmediated cGMP increase. 5) The role of basolaterally-secreted cGMP is speculated to modulate the proposed neuronal component of acute diarrheal disease, while apical secretion of cGMP may serve as a second messenger to either pathogenic or commensal inhabitants of the luminal space.

195 IN VITRO MODELING OF HUMAN ENTEROHEPATIC CIRCULATION USING STEM CELL-DERIVED ILEAL ENTEROIDS AND PRIMARY CULTURES OF HEPATOCYTES Sarah E. Blutt, James R. Broughman, Larry Vernetti, Mary Elizabeth M. Tessier, Sue E. Crawford, Xi-Lei Zeng, Tor C. Savidge, Karl-Dimiter Bissig, Jennifer D. Foulke-Abel, Nicholas C. Zachos, Olga Kovbasnjuk, D. Lansing Taylor, Mark Donowitz, Mary Estes Introduction. Enterohepatic circulation refers to the movement of bile acids, bilirubin, drugs, or other substances between the liver and the intestine. Bile acid synthesis and recycling is an important aspect of enterohepatic circulation that has not been able to be modeled in vitro. Methods. We functionally coupled human small intestinal enteroids (HIE), derived from stem cells isolated from human tissue, with an in vitro liver model consisting of primary human hepatocytes co-cultured with Kupffer, stellate, and endothelial cell lines. The HIEs, grown as polarized monolayers on Transwell™ permeable supports, were treated apically for 24 hr with bile acids (BA) or obeticholic acid (OCA) and bile acid transport and basolateral secretion of fibroblast growth factor (FGF19) from the cultures were evaluated. Basolateral fluid from ileal HIEs was added into the influx of the liver model and changes in transcripts and bile acid output were examined. Results. Apical BA exposure (24 hr) of ileal, but not duodenal or jejunal, HIEs induced basolateral secretion of FGF19 and transport of BA from the apical side of the HIE monolayer to the basolateral compartment. In addition, mRNA levels of FABP6 and SLC51alpha, which are transcriptional targets of BA and are important in BA transport across the epithelium, were upregulated in HIEs from all three intestinal segments. OCA treatment of ileal HIEs also increased levels of FGF19 in the basolateral media compared to untreated controls. Application of this basolateral media to the liver system decreased transcription of hepatic CYP7A1 mRNA and inhibited BA output. HepRG cells also responded similarly to the basolateral media of BA- or OCA-treated ileal HIEs with downregulation of CYP7A1 mRNA. Conclusion. These studies recapitulate key features of enterohepatic circulation including: 1) ileal apical uptake and basolateral secretion of BA, 2) BA- and OCA-induced ileal secretion of FGF19, and 3) downregulated liver CYP7A1 transcripts and bile acid secretion in hepatocytes treated with basolateral media from OCAtreated ileal HIEs. This in vitro model of the human enterohepatic circulation has the potential for understanding multi-organ interactions and physiology.

198 HUMAN PLURIPOTENT STEM CELL-DERIVED INTESTINAL ORGANOID MODEL FOR THE STUDY OF HUMAN RESPONSES TO INFECTION SHIGA TOXIN PRODUCING ESCHERICHIA COLI-INDUCED PATHOGENESIS Suman Pradhan, Sayali Karve, Alison A. Weiss

196 HUMAN-DERIVED GASTRIC CANCER ORGANOIDS SECRETE TUMOR ANTIGEN THAT ACTIVATES DENDRITIC CELLS AND SUBSEQUENT EXPRESSION OF PD-1 AND CTLA-4 ON CYTOTOXIC T LYMPHOCYTES Loryn L. Holokai, Nina Bertaux-Skeirik, Jayati Chakrabarti, Mark Wunderlich, Julie Chang, Emma L. Teal, Jennifer Hawkins, Nambirajan Sundaram, Maxime M. Mahe, Michael Helmrath, Syed Ahmad, James C. Mulloy, Yana Zavros

Background: Infection with Shiga toxin (Stx) producing Escherichia coli (STEC), such as O157:H7 can cause the potentially fatal complication hemolytic uremic syndrome. Currently only supportive therapy is available for treatment for this disease. Our knowledge of STEC is primarily based on data generated using human cancer cell lines that do not represent the normal human intestinal epithelium. Another serious limitation has been the lack of animal models. Thus, our objective has been to develop a human organoid-based model for STEC. Methods: Induced human intestinal organoids (iHIOs) were generated by in vitro differentiation of pluripotent stem cells and represent into differentiated human intestinal tissue. W,e and were used to compared examine intestinal responses to commensal E. coli and O157:H7 introduced into the lumen of iHIOsthe human specific pathogen, Shiga toxin (Stx) producing O157:H7. A iHIO/neutrophil co-culture system was also established to study the role of the immune response during infection. Results: Microinjected non-pCathogenic commensal E. coli replicated rapidly to high numbers, but were completely contained within the lumen and did not damage the iHIOorganoid. Pathogenic O157:H7 replicated equivalently, but severely damaged the epithelial layer, resulting in loss of epithelial barrier functionwith loss of the adherens junction protein, E-cadherin. Co-localization of O157:H7 colocalized with with cellular actin, consistent with intimate adherence. was observed at early time points. Different bacterial morphologies were also observedF. The commensals grew as short rods. Filamentous morphology was observed for the O157:H7 strain, consistent with activation of the bacterial SOS stress response. SOS was induced by in response to reactive oxygen species (ROS), and increased ROS was seen in response to O157:H7 infection. Transcriptional profiling (RNAseq) demonstrated that infection with either bacterial strain upregulated genes associated with gastrointestinal maturation. Infection with O157:H7 upregulated inflammatory responses, including interleukin 8 (IL-8). IL-8 is associated with neutrophil recruitment. Infection with O157:H7 resulted in recruitment of human neutrophils into the iHIO lumen tissue. Conclusion: The iHIO culture system represents a novel approach to study human-restricted enteric pathogens, including STEC using normal differentiated intestinal tissue in vitro.

Background: Tumors can evade immune surveillance by expressing ligands such as programmed cell death 1 (PDL-1) or B7-1/B7-2 which interact with PD-1 or CTLA-4, respectively, on CD8+ cytotoxic T lymphocytes (CTLs). These interactions inhibit CTL proliferation, survival and effector function subsequently leading to immune evasion and cancer persistence. Hypothesis: Tumor-secreted antigens activate a weakened anti-tumor immune response by inducing PD-1 and CTLA-4 expression on CTLs. Methods: Gastric organoids were generated from normal human stomachs (huFGO) and stomach tissue resected from the tumor of a patient with diffuse-type gastric cancer (huTGO). Conditioned media was collected from cultured huFGOCM or huTGOCM. Human peripheral blood mononuclear cells (PBMC) were used to isolate both dendritic cells (DCs) by culture and CD8+ CTLs by a CD8+ T Cell Enrichment Kit. DCs were pulsed with huFGOCM or huTGOCM for 24 hours and co-cultured with isolated CTLs for 72 hours. The expression of PD1, CTLA-4 and IL-2 in gated CD8+ cells were determined by flow cytometry. The "humanized" mouse model (huNRGS) was generated by engrafting NRGS mice with human umbilical cord blood. HuNRGS or nonhuman NRGS mice were used for orthortopic transplantation of huTGOs. Results: 1) HuTGOs express cancer stem cell marker CD44v9 and PDL-1: Early culture of huTGOs showed a similar phenotype to the culture of huFGOs. HuTGOs exhibited a heterogeneous population of cell clusters (proposed cancer stem cells) and organoids upon continued culture. Further passaging of huTGOs enriched for cell clusters that grew in an anchorage independent/soft agar assay and the expression of cancer stem cell marker CD44v9. CD44v9+ cells expressed PD-L1. 2) DCs pulsed with hTGOCM increase PD-1 and CTLA-4 expression on CTLs: Compared to the percent of CTLs expressing PD-1 and CTLA-4 co-cultured with huFGOCM-pulsed DC, huTGOCM-pulsed DCs induced expression of PD1 and CTLA4 on CTLs. 3) HuTGOs engraft within the huNRGS mouse stomach and contribute to the development of metaplasia: HuTGOs engrafted within the gastric epithelium of huNRGS mice and contributed to the development of metaplasia. Pending studies using CyTOF analysis will address the tumor-immune cell interactions in vivo. Immunofluorescence revealed infiltration of PD-1+ CTLs in human gastric cancers expressing CD44v9. Conclusion: HuTGOs secrete tumor antigens that induce DC activation. Activated DCs induce PD1 and CTLA-4 expression on CTLs that may result in inhibition of effector T cell function. The current study is the first report of an organoid-based approach for the study of the interaction between cancer cells and the immune microenvironment. This approach may be a preclinical organoid-based platform for personalized anticancer drug evaluation.

199 INTESTINE-CHIP: A NEW MODEL TO UNDERSTAND THE ROLE OF THE INTESTINAL EPITHELIUM IN IBD BY COMBINING MICROENGINEERING TECHNOLOGY AND IPSC-DERIVED HUMAN INTESTINAL ORGANOIDS Michael Workman, Elissa Troisi, S. Jordan Kerns, Geraldine A. Hamilton, Clive Svendsen, Stephan R. Targan, Robert J. Barrett Background: Inflammatory bowel disease (IBD) is a complex polygenic disorder characterized by chronic mucosal injury. It is believed to be caused by dysregulated immune responses to luminal microbes in genetically susceptible individuals. While some evidence suggests

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AGA Abstracts

AGA Abstracts

revealed a coordinated response to enterotoxigenic (ETEC) and enteropathogenic (EPEC) E. coli infections. Basolateral macrophages exhibited bacterial sensing and phagocytic activity in response to ETEC and EPEC exposure to the apical side of enteroid monolayers. Macrophages significantly reduced the presence of luminal ETEC and EPEC as early as 30 min post infection without increased expression of pro-inflammatory cytokines. CONCLUSIONS: In summary, we have established the first primary human macrophage-enteroid co-culture system, defined conditions that allow for a practical and reproducible culture model, and demonstrated its suitability to study gut physiology and host responses to enteric pathogens.