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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
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.
AGA Abstracts
that the intestinal epithelium is implicated, its precise role in IBD has remained elusive due a lack of suitable in vitro models. A major advance occurred with the development of intestinal organoids, whereby human intestinal organoids (HIOs) from control individuals or IBD patients, could be derived from induced pluripotent stem cells (iPSCs) or biopsy samples. However, in the context of IBD, this technology is challenging to use. Given that HIOs are polarized towards the lumen, studies examining intestinal permeability or bacterialepithelial interactions require access to the interior of the HIOs which is laborious and requires specialized equipment. In addition, studies examining epithelial-immune cell interactions are hampered as HIOs are embedded in a matrix. Methods: We have overcome such limitations by developing a novel Intestine-Chip. Initially, iPSCs were directed to form HIOs, then dissociated to a single cell suspension and finally seeded into a small microengineered Chip that is composed of two channels separated by a porous flexible membrane. The Chips are fluidic in nature, allowing continuous flow of medium in both channels and generation of in vivo relevant mechanical forces - critical for cell function. Results: We observed spontaneous formation of polarized villous-like structures that are similar to those found in the intestine. The presence of Paneth cells, goblet cells, enteroendocrine cells and enterocytes was confirmed by immunohistochemistry while in situ hybridization revealed the presence of LGR5+ cells. The secretion of antimicrobials from Paneth cells was detected by ELISA. The addition of IFNγ to the lower channel resulted in STAT1 phosphorylation and significant upregulation of the IFNγ responsive genes IDO1, GBP4 and GBP5. Interestingly, PLA2G2A and MUC4, two genes associated with Paneth cells and goblet cells respectively, were also upregulated. There was no upregulation of such genes in Caco2 cells. Conclusion: We have developed a system whereby iPSC-derived intestinal epithelium can be incorporated into a chip microenvironment and changes in gene expression and antimicrobial secretion can be measured. Given we have previously generated HIOs from iPSCs derived from lymphoblastoid cell lines (LCLs), we can now obtain genotyped IBD-LCLs stored by the MIRIAD Biobank in Cedars Sinai to generate intestinal epithelium containing genetic variants associated with IBD. This technology will allow us to assess how these variants influence the differentiation and functioning of this tissue and the response to various luminal microbes, immune cells and cytokines.
Proportion of Crohn's disease patients with a follow-up event, stratified by phenotype and treatment (Follow up event was defined as new IBD-related surgery, hospitalization, penetrating complication, steroid use, or change in biologic agent)
201 PATTERNS OF ANTI-TNF USE AND ASSOCIATED TREATMENT OUTCOMES IN INFLAMMATORY BOWEL DISEASE PATIENTS: RESULTS FROM AN ANALYSIS OF DUTCH HEALTH INSURANCE CLAIMS DATA Steven Bots, Daniel R. Hoekman, Marc A. Benninga, Cyriel Ponsioen, Hugo M. Smeets, Geert R. D'Haens, Mark Löwenberg Introduction: Patterns of anti-TNF use, associated treatment outcomes and drug costs have never been investigated in a large, real-life population of IBD patients. Methods: Health insurance claims data from 22,082 Dutch IBD patients were provided by Achmea Healthcare. Patients starting with anti-TNF treatment from January 2008 till December 2014 were studied. The primary analysis was time to anti-TNF discontinuation. Furthermore, time to anti-TNF treatment intensification, corticosteroid free survival and time to hospitalization were analyzed, as well as treatment regimens. Results: The proportion of infliximab (n=855) and adalimumab starters (n=1,199) who received intensified treatment increased over time (infliximab at 3 vs. 24 months: 22.2% vs. 33.6%, p=0.01; adalimumab at 3 vs. 24 months: 10.5% vs. 19.3%, p<0.001). Median time to anti-TNF discontinuation was 600 days (IQR 156-1693). Cessation of anti-TNF treatment was less common in Crohn's disease patients (HR 0.79, p=0.001) and in patients receiving intensified treatment regimens (HR 0.62, p= 0.001). Immunomodulator use was not related to longer drug survival (HR 0.99, p=0.617), but was significantly associated with longer time to corticosteroid use (HR 0.80, p=0.048). Hospitalization was significantly more common in Crohn's disease patients (HR 1.49, p= 0.011). Corticosteroid use was significantly lower in Crohn's disease patients (HR 0.57, p<0.001) and in patients using infliximab (HR 0.55, p<0.001). Conclusions: Discontinuation of anti-TNF therapy occurred earlier than previously reported and was associated with ulcerative colitis and non-intensified anti-TNF treatment regimens. Immunomodulator use at the start of anti-TNF treatment was associated with longer time to corticosteroid use, but not with longer drug survival.
Figure shows epithelial cells derived from human intestinal organoids forming villous-like structures in response to the continuous flow of media in the Intestine-Chip. CDX2 (red) and E-Cadherin (blue).
200 THE BENEFIT OF COMBINATION THERAPY DEPENDS ON DISEASE PHENOTYPE AND DURATION IN CROHN'S DISEASE: A PROSPECTIVE COHORT STUDY Ashwin Ananthakrishnan, Atsushi Sakuraba, Edward L. Barnes, Joel R. Pekow, Laura H. Raffals, Robert Sandler, Millie D. Long Introduction: In randomized controlled trials in early Crohn's disease (CD) and ulcerative colitis (UC), combination infliximab-azathioprine therapy is associated with greater rates of clinical remission and mucosal healing. Combination therapy may also be associated with higher risk of serious adverse events. . Importantly, the impact of combination therapy on disease-related morbidity in patients with established disease in the real-world setting remains to be well defined. Methods: We utilized data from a large, prospective cohort of CD and UC patients recruited from 7 referral centers (SHARE). At baseline, patients were classified as being on monotherapy with tumor necrosis factor antagonists (anti-TNF) (infliximab, adalimumab, certolizumab pegol, golimumab) or on combination therapy when anti-TNF was used in conjunction with a conventional immunomodulator (thiopurines, methotrexate). The primary outcome was a composite of new IBD-related surgery, hospitalizations, penetrating complications, need for steroids or new biologic therapy at 1 year. Results: The study included 707 patients with CD (391 (55%) monotherapy, 316 (45%) combination therapy) and 164 with UC (101 (62%) monotherapy, 63 (17%) combination therapy). Patients on combination therapy were similar to those on monotherapy in disease duration, phenotype (CD) or extent (UC) and were slightly more likely to have perianal involvement (29% vs. 21%). Similar proportions of patients on combination (61%) or monotherapy (63%) were on their first anti-TNF agent. 75% and 25% of combination therapy users were on thiopurines and methotrexate respectively. On multivariable analysis, combination therapy was not associated with reduction in the composite outcome in CD (OR 0.87, 95% CI 0.63 - 1.22) or UC (OR 1.45, 95% CI 0.63 - 3.38). However, the benefit in CD depended on disease phenotype (Figure). While no difference was noted in those with inflammatory B1 disease (OR 1.65, 95% CI 0.92 - 2.94), a significant reduction in the likelihood of the outcome was seen in those with structuring (B2) or penetrating disease phenotype (B3) (OR 0.58, 95% CI 0.37 - 0.90) (p-interaction 0.04). The strongest effects were seen in reduction of hospitalizations (p=0.01) and surgery (p=0.08). Among those with B2/B3 disease, a stronger effect was observed in those with disease duration < 5 years (OR 0.35, 95% CI 0.14 - 0.87) compared to those with a duration longer than 5 years (OR 0.75, 95% CI 0.45 - 1.27). Conclusion: The benefit of combination immunomodulator-biologic therapy is more striking in those with complicated Crohn's disease, particularly in those with disease duration less than 5 years.
AGA Abstracts
Univariable and multivariable Cox proportional hazards regression analysis of time to drug discontinuation
Kaplan-Meier curve of time to anti-TNF treatment discontinuation in Crohn's disease vs. ulcerative colitis patients
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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.
AGA Abstracts
that the intestinal epithelium is implicated, its precise role in IBD has remained elusive due a lack of suitable in vitro models. A major advance occurred with the development of intestinal organoids, whereby human intestinal organoids (HIOs) from control individuals or IBD patients, could be derived from induced pluripotent stem cells (iPSCs) or biopsy samples. However, in the context of IBD, this technology is challenging to use. Given that HIOs are polarized towards the lumen, studies examining intestinal permeability or bacterialepithelial interactions require access to the interior of the HIOs which is laborious and requires specialized equipment. In addition, studies examining epithelial-immune cell interactions are hampered as HIOs are embedded in a matrix. Methods: We have overcome such limitations by developing a novel Intestine-Chip. Initially, iPSCs were directed to form HIOs, then dissociated to a single cell suspension and finally seeded into a small microengineered Chip that is composed of two channels separated by a porous flexible membrane. The Chips are fluidic in nature, allowing continuous flow of medium in both channels and generation of in vivo relevant mechanical forces - critical for cell function. Results: We observed spontaneous formation of polarized villous-like structures that are similar to those found in the intestine. The presence of Paneth cells, goblet cells, enteroendocrine cells and enterocytes was confirmed by immunohistochemistry while in situ hybridization revealed the presence of LGR5+ cells. The secretion of antimicrobials from Paneth cells was detected by ELISA. The addition of IFNγ to the lower channel resulted in STAT1 phosphorylation and significant upregulation of the IFNγ responsive genes IDO1, GBP4 and GBP5. Interestingly, PLA2G2A and MUC4, two genes associated with Paneth cells and goblet cells respectively, were also upregulated. There was no upregulation of such genes in Caco2 cells. Conclusion: We have developed a system whereby iPSC-derived intestinal epithelium can be incorporated into a chip microenvironment and changes in gene expression and antimicrobial secretion can be measured. Given we have previously generated HIOs from iPSCs derived from lymphoblastoid cell lines (LCLs), we can now obtain genotyped IBD-LCLs stored by the MIRIAD Biobank in Cedars Sinai to generate intestinal epithelium containing genetic variants associated with IBD. This technology will allow us to assess how these variants influence the differentiation and functioning of this tissue and the response to various luminal microbes, immune cells and cytokines.
Proportion of Crohn's disease patients with a follow-up event, stratified by phenotype and treatment (Follow up event was defined as new IBD-related surgery, hospitalization, penetrating complication, steroid use, or change in biologic agent)
201 PATTERNS OF ANTI-TNF USE AND ASSOCIATED TREATMENT OUTCOMES IN INFLAMMATORY BOWEL DISEASE PATIENTS: RESULTS FROM AN ANALYSIS OF DUTCH HEALTH INSURANCE CLAIMS DATA Steven Bots, Daniel R. Hoekman, Marc A. Benninga, Cyriel Ponsioen, Hugo M. Smeets, Geert R. D'Haens, Mark Löwenberg Introduction: Patterns of anti-TNF use, associated treatment outcomes and drug costs have never been investigated in a large, real-life population of IBD patients. Methods: Health insurance claims data from 22,082 Dutch IBD patients were provided by Achmea Healthcare. Patients starting with anti-TNF treatment from January 2008 till December 2014 were studied. The primary analysis was time to anti-TNF discontinuation. Furthermore, time to anti-TNF treatment intensification, corticosteroid free survival and time to hospitalization were analyzed, as well as treatment regimens. Results: The proportion of infliximab (n=855) and adalimumab starters (n=1,199) who received intensified treatment increased over time (infliximab at 3 vs. 24 months: 22.2% vs. 33.6%, p=0.01; adalimumab at 3 vs. 24 months: 10.5% vs. 19.3%, p<0.001). Median time to anti-TNF discontinuation was 600 days (IQR 156-1693). Cessation of anti-TNF treatment was less common in Crohn's disease patients (HR 0.79, p=0.001) and in patients receiving intensified treatment regimens (HR 0.62, p= 0.001). Immunomodulator use was not related to longer drug survival (HR 0.99, p=0.617), but was significantly associated with longer time to corticosteroid use (HR 0.80, p=0.048). Hospitalization was significantly more common in Crohn's disease patients (HR 1.49, p= 0.011). Corticosteroid use was significantly lower in Crohn's disease patients (HR 0.57, p<0.001) and in patients using infliximab (HR 0.55, p<0.001). Conclusions: Discontinuation of anti-TNF therapy occurred earlier than previously reported and was associated with ulcerative colitis and non-intensified anti-TNF treatment regimens. Immunomodulator use at the start of anti-TNF treatment was associated with longer time to corticosteroid use, but not with longer drug survival.
Figure shows epithelial cells derived from human intestinal organoids forming villous-like structures in response to the continuous flow of media in the Intestine-Chip. CDX2 (red) and E-Cadherin (blue).
200 THE BENEFIT OF COMBINATION THERAPY DEPENDS ON DISEASE PHENOTYPE AND DURATION IN CROHN'S DISEASE: A PROSPECTIVE COHORT STUDY Ashwin Ananthakrishnan, Atsushi Sakuraba, Edward L. Barnes, Joel R. Pekow, Laura H. Raffals, Robert Sandler, Millie D. Long Introduction: In randomized controlled trials in early Crohn's disease (CD) and ulcerative colitis (UC), combination infliximab-azathioprine therapy is associated with greater rates of clinical remission and mucosal healing. Combination therapy may also be associated with higher risk of serious adverse events. . Importantly, the impact of combination therapy on disease-related morbidity in patients with established disease in the real-world setting remains to be well defined. Methods: We utilized data from a large, prospective cohort of CD and UC patients recruited from 7 referral centers (SHARE). At baseline, patients were classified as being on monotherapy with tumor necrosis factor antagonists (anti-TNF) (infliximab, adalimumab, certolizumab pegol, golimumab) or on combination therapy when anti-TNF was used in conjunction with a conventional immunomodulator (thiopurines, methotrexate). The primary outcome was a composite of new IBD-related surgery, hospitalizations, penetrating complications, need for steroids or new biologic therapy at 1 year. Results: The study included 707 patients with CD (391 (55%) monotherapy, 316 (45%) combination therapy) and 164 with UC (101 (62%) monotherapy, 63 (17%) combination therapy). Patients on combination therapy were similar to those on monotherapy in disease duration, phenotype (CD) or extent (UC) and were slightly more likely to have perianal involvement (29% vs. 21%). Similar proportions of patients on combination (61%) or monotherapy (63%) were on their first anti-TNF agent. 75% and 25% of combination therapy users were on thiopurines and methotrexate respectively. On multivariable analysis, combination therapy was not associated with reduction in the composite outcome in CD (OR 0.87, 95% CI 0.63 - 1.22) or UC (OR 1.45, 95% CI 0.63 - 3.38). However, the benefit in CD depended on disease phenotype (Figure). While no difference was noted in those with inflammatory B1 disease (OR 1.65, 95% CI 0.92 - 2.94), a significant reduction in the likelihood of the outcome was seen in those with structuring (B2) or penetrating disease phenotype (B3) (OR 0.58, 95% CI 0.37 - 0.90) (p-interaction 0.04). The strongest effects were seen in reduction of hospitalizations (p=0.01) and surgery (p=0.08). Among those with B2/B3 disease, a stronger effect was observed in those with disease duration < 5 years (OR 0.35, 95% CI 0.14 - 0.87) compared to those with a duration longer than 5 years (OR 0.75, 95% CI 0.45 - 1.27). Conclusion: The benefit of combination immunomodulator-biologic therapy is more striking in those with complicated Crohn's disease, particularly in those with disease duration less than 5 years.
AGA Abstracts
Univariable and multivariable Cox proportional hazards regression analysis of time to drug discontinuation
Kaplan-Meier curve of time to anti-TNF treatment discontinuation in Crohn's disease vs. ulcerative colitis patients
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