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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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BACKGROUND & AIMS: Gene expression patterns of CD8+ T cells have been reported to correlate with clinical outcomes of adults with inflammatory bowel diseases (IBD). We aimed to validate these findings in independent patient cohorts. METHODS: We obtained peripheral blood samples from 112 children with a new diagnosis of IBD (71 with Crohn's disease and 41 with ulcerative colitis) and 19 children without IBD (controls) and recorded medical information on disease activity and outcomes. CD8+ T cells were isolated from blood samples by magnetic bead sorting at the point of diagnosis and during the course of disease. Genome-wide transcription (n = 192) and DNA methylation (n = 66) profiles were generated using Affymetrix and Illumina arrays, respectively. Publicly available transcriptomes and DNA methylomes of CD8+ T cells from 3 adult patient cohorts with and without IBD were included in data analyses. RESULTS: Previously reported CD8+ T-cell prognostic expression and exhaustion signatures were only found in the original adult IBD patient cohort. These signatures could not be detected in either a pediatric or a second adult IBD cohort. In contrast, an association between CD8+ T-cell gene expression with age and sex was detected across all 3 cohorts. CD8+ gene transcription was clearly associated with IBD in the 2 cohorts that included non-IBD controls. Lastly, DNA methylation profiles of CD8+ T cells from children with Crohn's disease correlated with age but not with disease outcome. CONCLUSIONS: We were unable to validate previously reported findings of an association between CD8+ T-cell gene transcription and disease outcome in IBD. Our findings reveal the challenges of developing prognostic biomarkers for patients with IBD and the importance of their validation in large, independent cohorts before clinical application.