Assessing DNA methylation in the developing human intestinal epithelium: potential link to inflammatory bowel disease

Abstract An in vitro model of intestine epithelium with an immune compartment was bioengineered to mimic immunologic responses seen in inflammatory bowel disease [1]. While aspects of intestinal immunity can be modeled in transwells and 2D culture systems, 3D tissue models improve physiological relevance by providing a 3D substrate which enable migration of macrophages towards the epithelium. An intestinal epithelium comprised of non-transformed human colon organoid cells and a subepithelial layer laden with monocyte-derived macrophages was bioengineered to mimic native intestinal mucosa cell organization using spongy silk scaffolds. Confluent epithelial monolayers with microvilli, a mucus layer, and infiltration of macrophages to the basal side of the epithelium were observed. Inflammation, induced by E. coli O111:B4 lipopolysaccharide and interferon γ resulted in morphology changes to the epithelium, resulting in ball-like structures, decreased epithelial coverage, and migration of macrophages to the epithelium. Analysis of cytokines present in the inflamed tissue model demonstrated significantly upregulated secretion of pro-inflammatory cytokines associated with active inflammatory bowel disease, including CXCL10, IL-1β, IL-6, MCP-2, and MIP-1β. The macrophage layer enhanced epithelial and biochemical responses to inflammatory stimuli, and this new tissue system may be useful to study and develop potential therapies for inflammatory bowel disease. References: 6 Roh, T.T., et al., 3D bioengineered tissue model of the large intestine to study inflammatory bowel disease. Biomaterials, 2019: p. 119517. 7 In, J., et al., Enterohemorrhagic Escherichia coli reduce mucus and intermicrovillar bridges in human stem cell-derived colonoids. Cellular and molecular gastroenterology and hepatology, 2015. 2(1): p. 48–62.e3. 8 Chen, Y., et al., In vitro enteroid-derived three-dimensional tissue model of human small intestinal epithelium with innate immune responses. PLoS ONE, 2017. 12(11): p. e0187880. Colonoid and macrophage cultivation scheme in the 3D bilayer system. (A) Human monocytes were isolated from whole blood and human colonoids from large intestine biopsies were cultured according to established protocols [2]. (B) Cell suspensions of colonoids were seeded on the film surface on the inner silk scaffold and monocyte-derived macrophages were seeded throughout the porous outer silk scaffold using established protocols [3]. (C) The model is cultured for 3 weeks total with 2 weeks in High WNT media and 1 week in differentiation media based on established protocol. Colonoids are present in the model throughout the 3 week culture time. 2 sets of macrophages are added with the first set added after the first week of culture and the second set replacing the first set after the second week.

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Intestinal Epithelium in Inflammatory Bowel Disease

Frontiers in Medicine ◽

10.3389/fmed.2014.00024 ◽

2014 ◽

Vol 1 ◽

Cited By ~ 48

Author(s):

Mehmet Coskun

Keyword(s):

Inflammatory Bowel Disease ◽

Intestinal Epithelium ◽

Bowel Disease ◽

Inflammatory Bowel

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Epigenetic Modifications of the Nuclear Factor Kappa B Signalling Pathway and its Impact on Inflammatory Bowel Disease

Current Pharmaceutical Design ◽

10.2174/1381612827666210218141847 ◽

2021 ◽

Vol 27 ◽

Author(s):

Stamatia Papoutsopoulou ◽

Barry J. Campbell

Keyword(s):

Dna Methylation ◽

Inflammatory Bowel Disease ◽

Bowel Disease ◽

Nuclear Factor Kappa B ◽

Intestinal Inflammation ◽

Alternative Pathway ◽

Current Evidence ◽

Nuclear Factor ◽

Nuclear Factor Kappa ◽

Inflammatory Bowel

Background: Inflammatory bowel disease (IBD) is a multifactorial condition influenced by the immune system, the intestinal microbiota, environmental factors, genetic and epigenetic factors. Genetic- and environment-induced dysregulation of the Nuclear Factor-kappa B (NF-κB) transcription factor pathway has been linked to IBD pathogenesis. Objective: To assess the current evidence in relation to the contribution of the classical and alternative NF-κB pathways in IBD and to discuss the epigenetic mechanisms that impact on NF-κB function. Methods: A Medline search for ‘NF-kappaB/NF-κB’, in combination with terms including ‘inflammatory bowel disease/IBD’, 'intestinal inflammation', ‘Crohn's disease’, ‘ulcerative colitis’, 'colitis'; ‘epigenetics’, ‘DNA methylation’, ‘histones’, ‘microRNAs/miRNAs’ and ‘short non-coding/long non-coding RNAs’ was performed. Results: Both NF-κB pathways contribute to the chronic inflammation underlying IBD by regulating the inflammatory immune responses and homeostasis of the intestinal epithelium (classical pathway) or regulating bowel inflammation and epithelial microfold (M) cell function (alternative pathway). DNA methylation is a common epigenetic modification in intestinal inflammation, including NFKB1 and RELA loci. Conversely, little is understood regarding epigenetic effects on genes encoding other NF-κB subunits, particularly those of the alternative pathway, and in the context of IBD. However, NF-κB interaction with chromatin modifiers is also seen to be an essential mechanism of regulation of downstream target genes relevant to NF-κB-mediated inflammatory responses. Conclusion: Further research is clearly warranted in this area, as understanding the cell-specific regulation of the NF-κB pathways will bring researchers into a position to achieve more efficient stratification of IBD patients, and more targeted and effective choice of treatment.

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DNA Methylation Profiling in Inflammatory Bowel Disease Provides New Insights into Disease Pathogenesis

Journal of Crohn s and Colitis ◽

10.1093/ecco-jcc/jjv176 ◽

2015 ◽

Vol 10 (1) ◽

pp. 77-86 ◽

Cited By ~ 58

Author(s):

Edel McDermott ◽

Elizabeth J. Ryan ◽

Miriam Tosetto ◽

David Gibson ◽

Joe Burrage ◽

...

Keyword(s):

Dna Methylation ◽

Inflammatory Bowel Disease ◽

Bowel Disease ◽

Disease Pathogenesis ◽

Dna Methylation Profiling ◽

Inflammatory Bowel ◽

Methylation Profiling

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OC-010 Epigenome-wide dna methylation profiling in inflammatory bowel disease

Gut ◽

10.1136/gutjnl-2015-309861.10 ◽

2015 ◽

Vol 64 (Suppl 1) ◽

pp. A5.2-A6

Author(s):

NT Ventham ◽

NA Kennedy ◽

AT Adams ◽

R Kalla ◽

KR O’Leary ◽

...

Keyword(s):

Dna Methylation ◽

Inflammatory Bowel Disease ◽

Bowel Disease ◽

Dna Methylation Profiling ◽

Inflammatory Bowel ◽

Methylation Profiling

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Differential Alteration in Intestinal Epithelial Cell Expression of Toll-Like Receptor 3 (TLR3) and TLR4 in Inflammatory Bowel Disease

Infection and Immunity ◽

10.1128/iai.68.12.7010-7017.2000 ◽

2000 ◽

Vol 68 (12) ◽

pp. 7010-7017 ◽

Cited By ~ 883

Author(s):

Elke Cario ◽

Daniel K. Podolsky

Keyword(s):

Inflammatory Bowel Disease ◽

Epithelial Cell ◽

Intestinal Epithelium ◽

Bowel Disease ◽

Intestinal Epithelial Cell ◽

Intestinal Epithelial Cells ◽

Intestinal Epithelial ◽

Innate Response ◽

Response System ◽

Inflammatory Bowel

ABSTRACT Initiation and perpetuation of the inflammatory intestinal responses in inflammatory bowel disease (IBD) may result from an exaggerated host defense reaction of the intestinal epithelium to endogenous lumenal bacterial flora. Intestinal epithelial cell lines constitutively express several functional Toll-like receptors (TLRs) which appear to be key regulators of the innate response system. The aim of this study was to characterize the expression pattern of TLR2, TLR3, TLR4, and TLR5 in primary intestinal epithelial cells from patients with IBD. Small intestinal and colonic biopsy specimens were collected from patients with IBD (Crohn's disease [CD], ulcerative colitis [UC]) and controls. Non-IBD specimens were assessed by immunofluorescence histochemistry using polyclonal antibodies specific for TLR2, TLR3, TLR4, and TLR5. Primary intestinal epithelial cells (IEC) of normal mucosa constitutively expressed TLR3 and TLR5, while TLR2 and TLR4 were only barely detectable. In active IBD, the expression of TLR3 and TLR4 was differentially modulated in the intestinal epithelium. TLR3 was significantly downregulated in IEC in active CD but not in UC. In contrast, TLR4 was strongly upregulated in both UC and CD. TLR2 and TLR5 expression remained unchanged in IBD. These data suggest that IBD may be associated with distinctive changes in selective TLR expression in the intestinal epithelium, implying that alterations in the innate response system may contribute to the pathogenesis of these disorders.

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