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Sa1825 Intestinal Organoids Derived From Patients With Inflammatory Bowel Disease Show Unaltered Transcriptional Profiles When Compared to Healthy Controls
Sa1825
in human intestinal organoids of normal and IBD patients. Methods: Following differentiation, we stimulated organoids overnight with recombinant cytokines IL-1b, IL-17, IL-22, TNF, and IL-13 and measured changes in cytokine-inducible genes (DMBT1, REG3A, CXCL1, SAA1/2, CCL26, POSTN). We assessed the effects of the corticosteroid dexamethasone in combination with IL-22. We also measured cytokine expression and cytokine-inducible gene expression in tissue biopsies from non-IBD and IBD patients. Results: Organoids responded to cytokine stimulation with altered gene expression. IL-22 inducible genes including DMBT1, CXCL1, and SAA1/2, but not REG3A were found to be elevated following IL-22 stimulation in both DVT and UC patients. Co-treatment with dexamethasone did not significantly alter expression of these genes. IL-17 stimulation increased expression of CXCL1 and SAA1/2. CCL26, but not POSTN, was induced in IL-13-stimulated organoids. IL-13 induction of gene expression was relatively specific, with little induction of CCL26 by other cytokines. However, induction of CXCL1 and SAA1/2 was observed with TNF or IL-17 treatment. We evaluated baseline differences in gene expression in organoids, and observed upregulation of cytokines, and cytokine-inducible genes in IBD samples in comparison to controls. Conclusion: Primary human organoids represent a valuable resource for assessing changes in gene expression following specific stimulation. We observed relatively specific responses in some cytokine-inducible genes in vitro (DMBT1, CCL26) with little effect of steroid treatment on expression levels. Other genes (CXCL1, SAA1/2) were upregulated by more than one cytokine. All of these genes were upregulated in IBD samples, which may reflect cytokine activity during active disease.
AGA Abstracts
Intestinal Organoids Derived From Patients With Inflammatory Bowel Disease Show Unaltered Transcriptional Profiles When Compared to Healthy Controls Manuel Noben, Nikolai Hendriks, Gert Van Assche, Severine Vermeire, Catherine Verfaillie, Marc Ferrante Background Various mechanisms contribute to the pathogenesis of inflammatory bowel diseases (IBD), including microbial dysbiosis and defects in epithelial barrier, Paneth cell or goblet cell function. The epithelium is constantly renewed by intestinal stem cells (ISCs) located at the bottom of the crypts. The ex vivo ISC-containing organoids may be a suitable model to investigate IBD pathogenesis. Methods We evaluated if the organoid forming capacity of colonic crypts, as well as organoid transcriptional profiles, from IBD patients differ from that of healthy controls (HC). Colonic mucosal biopsies from 10 HC, 11 ulcerative colitis (UC) patients, and 11 Crohn's disease (CD) patients were used to culture organoids as previously described. After 2 passages, organoids were differentiated for 4 days by withdrawal of Wnt3a, nicotinamide and p38-inhibitor from the medium. RNA and histology samples were processed and analyzed using qPCR and immunohistochemistry (IHC) for ISCs and differentiated cell types as well as proliferation and apoptosis (Ki-67 and cleaved caspase 3 staining). We also investigated how inflammation (IL8, TNF a, CXCL3, among others) evolved when organoids were established from macroscopic inflamed areas. We included 3 patients per group (HC, UC, CD) and cultured organoids from crypts derived from both macroscopic inflamed and non-inflamed tissue. RNA was isolated from fresh biopsies and 1 week old organoids derived from the same tissue. Results There was no difference in initial organoid forming capacity between crypts of controls and IBD patients (HC 77%, UC 79%, CD 80% P=0.51). Additionally, no changes in expression of the ISCmarker Lgr5 were found. The expression of Hath1, a positive regulator of goblet cell differentiation, was decreased in UC and CD compared to HC, both in differentiated as well as undifferentiated conditions. Also, chromogranin A expression in UC patients was decreased under differentiation, while in differentiated organoids from CD patients a decrease in mucin2 expression was observed. Moreover, preliminary data showed an unexpected increase in IL8 expression when both macroscopic and non-inflamed tissue was cultured as organoids, while expression of CXCL3 was down-regulated. Conclusions Our data shows that intestinal crypts isolated from IBD patients form organoids as efficient as crypts from healthy controls. Gene expression of markers of stemness and differentiation showed only subtle differences, and the biological implications remain to be clarified via immunohistochemistry. We are validating these results by expanding the cohort and testing more genes. P-values from organoid differentiation experiment
Sa1827 Molecular Landscape of Ulcerative Colitis and Crohn's Disease is Conserved Vojislav Jovanovic, Jeffery Venner, Jessica Chang, Philip Halloran, Richard N. Fedorak, Brendan P. Halloran INTRODUCTION: While disease-specific differences between IBD phenotypes are important, it is also of interest to see the conserved elements that reflect the response to injury shared by the phenotypes. METHODS: To map the elements conserved between Ulcerative Colitis (UC) and ileal Crohn's Disease (CD) we used microarrays to study the molecular landscape of 63 UC biopsies compared to 16 control colon biopsies, and 37 ileal CD biopsies compared to 7 control ileal biopsies. These comparisons were expressed as "molecular landscapes" using volcano plots of molecular association strength via p-value (x-axis) versus fold change (y-axis). The landscape of UC (Fig 1) was compared to that of ileal CD (Fig 2) for all 13709 interquartile-range filtered probe sets. We labeled transcripts of interest, including TNFalpha, calprotectin (S100A8 and S100A9), TNFalpha-inducible transcripts, inflammasomeassociated transcripts, IFNG-inducible transcripts, transcripts representing the response to injury (increased in UC and CD), and transcripts decreased in injured tissue (conserved epithelial genes associated with parenchymal function and metabolism). RESULTS: There was striking conservation between the molecular landscape of the two disease processes. In both UC and CD, TNF-alpha was interestingly only mildly increased in UC (Fold change= 1.2, P=NS) and CD (Fold change=2.4, P=0.02) compared to controls; however calprotectin (S100A8 and S100A9) was strongly induced in both UC (P=0.0006) and ileal CD (P=0.0001) but the fold change increase in CD was higher than that induced in UC (14x vs 4x). TNFalpha-inducible and inflammasome-associated transcripts were highly conserved across both UC and ileal CD. As expected, expression of inflammasome transcript NOD2 was only associated with CD (Fold change=2.7, P=0.003), but not in UC (P=NS). Epithelial transcripts were variably downregulated in both diseases, indicating the stereotyped dedifferentiation of the parenchyma. Analyses of specific differences between UC and ileal CD and of the most significantly up- and downregulated signals are currently being undertaken. CONCLUSION: We conclude that although one might expect UC and ileal CD would have different inflammatory profiles as they present with markedly different phenotypes and in different epithelia, the large-scale molecular changes are strikingly conserved. Calprotectin expression was high in UC and CD in keeping with its current use a fecal biomarker in both diseases.
Expression data from colonic organoids cultured from crypts from IBD patients and controls. Upper panel data from organoids in expansion medium, lower panel after 4 days in differentiation medium. Figure 1. Molecular landscape of UC, represented by volcano plot. Sa1826 Identification of Gene Signatures in Human Intestinal Organoids for Biomarker Analyses Deepti Nagarkar, Paolo Manzanillo, Ryan Ichikawa, Julie Rae, Luz Orozco, Jason Hackney, Ann Herman, Mary Keir Introduction: Inflammatory bowel disease (IBD) includes Crohn's disease (CD) and ulcerative colitis (UC) and is characterized by increased inflammation in the gut, impaired mucosal barrier function, and microbial dysbiosis. Inflammatory and regulatory cytokines are dysregulated in IBD and can directly affect the epithelial mucosa. Cytokine effects on primary epithelial cells can be modeled in vitro using organoid culture. We evaluated cytokineinducible gene-expression at baseline and following treatment with IL-22, IL-17, and IL-13
AGA Abstracts
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in human intestinal organoids of normal and IBD patients. Methods: Following differentiation, we stimulated organoids overnight with recombinant cytokines IL-1b, IL-17, IL-22, TNF, and IL-13 and measured changes in cytokine-inducible genes (DMBT1, REG3A, CXCL1, SAA1/2, CCL26, POSTN). We assessed the effects of the corticosteroid dexamethasone in combination with IL-22. We also measured cytokine expression and cytokine-inducible gene expression in tissue biopsies from non-IBD and IBD patients. Results: Organoids responded to cytokine stimulation with altered gene expression. IL-22 inducible genes including DMBT1, CXCL1, and SAA1/2, but not REG3A were found to be elevated following IL-22 stimulation in both DVT and UC patients. Co-treatment with dexamethasone did not significantly alter expression of these genes. IL-17 stimulation increased expression of CXCL1 and SAA1/2. CCL26, but not POSTN, was induced in IL-13-stimulated organoids. IL-13 induction of gene expression was relatively specific, with little induction of CCL26 by other cytokines. However, induction of CXCL1 and SAA1/2 was observed with TNF or IL-17 treatment. We evaluated baseline differences in gene expression in organoids, and observed upregulation of cytokines, and cytokine-inducible genes in IBD samples in comparison to controls. Conclusion: Primary human organoids represent a valuable resource for assessing changes in gene expression following specific stimulation. We observed relatively specific responses in some cytokine-inducible genes in vitro (DMBT1, CCL26) with little effect of steroid treatment on expression levels. Other genes (CXCL1, SAA1/2) were upregulated by more than one cytokine. All of these genes were upregulated in IBD samples, which may reflect cytokine activity during active disease.
AGA Abstracts
Intestinal Organoids Derived From Patients With Inflammatory Bowel Disease Show Unaltered Transcriptional Profiles When Compared to Healthy Controls Manuel Noben, Nikolai Hendriks, Gert Van Assche, Severine Vermeire, Catherine Verfaillie, Marc Ferrante Background Various mechanisms contribute to the pathogenesis of inflammatory bowel diseases (IBD), including microbial dysbiosis and defects in epithelial barrier, Paneth cell or goblet cell function. The epithelium is constantly renewed by intestinal stem cells (ISCs) located at the bottom of the crypts. The ex vivo ISC-containing organoids may be a suitable model to investigate IBD pathogenesis. Methods We evaluated if the organoid forming capacity of colonic crypts, as well as organoid transcriptional profiles, from IBD patients differ from that of healthy controls (HC). Colonic mucosal biopsies from 10 HC, 11 ulcerative colitis (UC) patients, and 11 Crohn's disease (CD) patients were used to culture organoids as previously described. After 2 passages, organoids were differentiated for 4 days by withdrawal of Wnt3a, nicotinamide and p38-inhibitor from the medium. RNA and histology samples were processed and analyzed using qPCR and immunohistochemistry (IHC) for ISCs and differentiated cell types as well as proliferation and apoptosis (Ki-67 and cleaved caspase 3 staining). We also investigated how inflammation (IL8, TNF a, CXCL3, among others) evolved when organoids were established from macroscopic inflamed areas. We included 3 patients per group (HC, UC, CD) and cultured organoids from crypts derived from both macroscopic inflamed and non-inflamed tissue. RNA was isolated from fresh biopsies and 1 week old organoids derived from the same tissue. Results There was no difference in initial organoid forming capacity between crypts of controls and IBD patients (HC 77%, UC 79%, CD 80% P=0.51). Additionally, no changes in expression of the ISCmarker Lgr5 were found. The expression of Hath1, a positive regulator of goblet cell differentiation, was decreased in UC and CD compared to HC, both in differentiated as well as undifferentiated conditions. Also, chromogranin A expression in UC patients was decreased under differentiation, while in differentiated organoids from CD patients a decrease in mucin2 expression was observed. Moreover, preliminary data showed an unexpected increase in IL8 expression when both macroscopic and non-inflamed tissue was cultured as organoids, while expression of CXCL3 was down-regulated. Conclusions Our data shows that intestinal crypts isolated from IBD patients form organoids as efficient as crypts from healthy controls. Gene expression of markers of stemness and differentiation showed only subtle differences, and the biological implications remain to be clarified via immunohistochemistry. We are validating these results by expanding the cohort and testing more genes. P-values from organoid differentiation experiment
Sa1827 Molecular Landscape of Ulcerative Colitis and Crohn's Disease is Conserved Vojislav Jovanovic, Jeffery Venner, Jessica Chang, Philip Halloran, Richard N. Fedorak, Brendan P. Halloran INTRODUCTION: While disease-specific differences between IBD phenotypes are important, it is also of interest to see the conserved elements that reflect the response to injury shared by the phenotypes. METHODS: To map the elements conserved between Ulcerative Colitis (UC) and ileal Crohn's Disease (CD) we used microarrays to study the molecular landscape of 63 UC biopsies compared to 16 control colon biopsies, and 37 ileal CD biopsies compared to 7 control ileal biopsies. These comparisons were expressed as "molecular landscapes" using volcano plots of molecular association strength via p-value (x-axis) versus fold change (y-axis). The landscape of UC (Fig 1) was compared to that of ileal CD (Fig 2) for all 13709 interquartile-range filtered probe sets. We labeled transcripts of interest, including TNFalpha, calprotectin (S100A8 and S100A9), TNFalpha-inducible transcripts, inflammasomeassociated transcripts, IFNG-inducible transcripts, transcripts representing the response to injury (increased in UC and CD), and transcripts decreased in injured tissue (conserved epithelial genes associated with parenchymal function and metabolism). RESULTS: There was striking conservation between the molecular landscape of the two disease processes. In both UC and CD, TNF-alpha was interestingly only mildly increased in UC (Fold change= 1.2, P=NS) and CD (Fold change=2.4, P=0.02) compared to controls; however calprotectin (S100A8 and S100A9) was strongly induced in both UC (P=0.0006) and ileal CD (P=0.0001) but the fold change increase in CD was higher than that induced in UC (14x vs 4x). TNFalpha-inducible and inflammasome-associated transcripts were highly conserved across both UC and ileal CD. As expected, expression of inflammasome transcript NOD2 was only associated with CD (Fold change=2.7, P=0.003), but not in UC (P=NS). Epithelial transcripts were variably downregulated in both diseases, indicating the stereotyped dedifferentiation of the parenchyma. Analyses of specific differences between UC and ileal CD and of the most significantly up- and downregulated signals are currently being undertaken. CONCLUSION: We conclude that although one might expect UC and ileal CD would have different inflammatory profiles as they present with markedly different phenotypes and in different epithelia, the large-scale molecular changes are strikingly conserved. Calprotectin expression was high in UC and CD in keeping with its current use a fecal biomarker in both diseases.
Expression data from colonic organoids cultured from crypts from IBD patients and controls. Upper panel data from organoids in expansion medium, lower panel after 4 days in differentiation medium. Figure 1. Molecular landscape of UC, represented by volcano plot. Sa1826 Identification of Gene Signatures in Human Intestinal Organoids for Biomarker Analyses Deepti Nagarkar, Paolo Manzanillo, Ryan Ichikawa, Julie Rae, Luz Orozco, Jason Hackney, Ann Herman, Mary Keir Introduction: Inflammatory bowel disease (IBD) includes Crohn's disease (CD) and ulcerative colitis (UC) and is characterized by increased inflammation in the gut, impaired mucosal barrier function, and microbial dysbiosis. Inflammatory and regulatory cytokines are dysregulated in IBD and can directly affect the epithelial mucosa. Cytokine effects on primary epithelial cells can be modeled in vitro using organoid culture. We evaluated cytokineinducible gene-expression at baseline and following treatment with IL-22, IL-17, and IL-13
AGA Abstracts
X : 10052$CH01 03-28-16 00:57:27 PDFd : 10052B : e
S-374
Page 374