Patient-derived organoid biobank identifies epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in severe Crohn's Disease.
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OBJECTIVE: Epigenetic mechanisms, including DNA methylation (DNAm), have been proposed to play a key role in Crohn's disease (CD) pathogenesis. However, the specific cell types and pathways affected as well as their potential impact on disease phenotype and outcome remain unknown. We set out to investigate the role of intestinal epithelial DNAm in CD pathogenesis. DESIGN: We generated 312 intestinal epithelial organoids (IEOs) from mucosal biopsies of 168 patients with CD (n=72), UC (n=23) and healthy controls (n=73). We performed genome-wide molecular profiling including DNAm, bulk as well as single-cell RNA sequencing. Organoids were subjected to gene editing and the functional consequences of DNAm changes evaluated using an organoid-lymphocyte coculture and a nucleotide-binding oligomerisation domain, leucine-rich repeat and CARD domain containing 5 (NLRC5) dextran sulphate sodium (DSS) colitis knock-out mouse model. RESULTS: We identified highly stable, CD-associated loss of DNAm at major histocompatibility complex (MHC) class 1 loci including NLRC5 and cognate gene upregulation. Single-cell RNA sequencing of primary mucosal tissue and IEOs confirmed the role of NLRC5 as transcriptional transactivator in the intestinal epithelium. Increased mucosal MHC-I and NLRC5 expression in adult and paediatric patients with CD was validated in additional cohorts and the functional role of MHC-I highlighted by demonstrating a relative protection from DSS-mediated mucosal inflammation in NLRC5-deficient mice. MHC-I DNAm in IEOs showed a significant correlation with CD disease phenotype and outcomes. Application of machine learning approaches enabled the development of a disease prognostic epigenetic molecular signature. CONCLUSIONS: Our study has identified epigenetically regulated intestinal epithelial MHC-I as a novel mechanism in CD pathogenesis.
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Acknowledgements: The authors would like to thank Prof Gillian Griffith (Cambridge Institute for Medical Research, CIMR, Cambridge) for providing helpful guidance and access to tissue from OTI mice. We thank the University of Lausanne for sharing transgenic mice and Dr. A. Zenobi (IRB Bellinzona) who helped with the mouse shipment’s organization. We would like also to acknowledge Sophie Pritchard (Cellular Generation and Phenotyping core facility at the Wellcome Sanger Institute) and Dr. Heather Zecchini (CRUK Cambridge Institute Light Microscopy facility) for helping us with RNA scope and spinning disk microscopy image acquisition and analysis respectively. Furthermore, we thank Prof Louise Boyle (Department of Pathology, University of Cambridge) for advice and constructive discussions. The illustrative graphics of experiments were created with BioRender.com.
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1468-3288
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Cancer Research UK (A25117)
Wellcome Trust (220268/Z/20/Z)