Spotlight: Epigenetic control of epithelial MHC-I in Crohn’s disease

Introduction

Crohn’s disease (CD) is characterised by relapsing inflammation that recurs in the same regional locations within the intestine. This suggests that permanent or long-lasting, molecular changes are occurring at the cellular level. Epigenetic modifications such as DNA methylation (DNAm) have been implicated in CD, but the precise epithelial mechanisms are unclear.

For this research, the authors have generated a large biobank of patient-derived intestinal organoids and identified a stable, Crohn’s disease-associated loss of DNAm at MHC-I (Major Histocompatibility complex) pathway genes, most notably at the promoter of the gene coding for the pattern recognition receptor, NLRC5. The NLRC5 protein is a key regulator of MHC class I genes (HLA-A, HLA-B and HLA-C).

The epigenetic dysregulation of NLRC5 resulted in enhanced MHC-I expression and altered crosstalk with immune cells. Functional studies in organoids and mouse models demonstrated that NLRC5-driven epithelial MHC-I contributes to mucosal inflammation.

Main Points

• A biobank of 312 intestinal epithelial organoid (IEO) lines was established from 168 patients (72 Crohn’s, 23 Ulcerative Colitis, 73 healthy).
• Genome-wide DNAm profiling identified stable hypomethylation at MHC-I loci (NLRC5, TAP1/2, HLA genes) in CD organoids.
• These changes remained across organoid passages and were independent of inflammatory stimuli confirming that they reflect heritable epithelial alterations.
• Analysis of patient intestinal epithelial tissue also demonstrated loss of DNAm and increased transcription of NLRC5 and MHC-I genes in CD epithelium.
• Organoids from CD patients showed higher NLRC5 expression and stronger induction of MHC-I after IFNγ stimulation.
• CRISPR-Cas9 knockout and inducible overexpression studies in human intestinal epithelial organoids (IEO’s) established NLRC5 as a transcriptional activator of MHC-I in intestinal epithelial cells. Overexpression was found to amplify IFNγ-driven MHC-I expression, while NLRC5 deletion led a reduced induction of expression.
• The same results were confirmed in organoids grown from NLRC5-deficient mice, thus demonstrating that NLRC5 is capable of upregulating intestinal epithelial MHC-I and increases sensitivity to IFNγ.
• Single-cell RNA sequencing of intestinal biopsies demonstrated differential MHC-I expression with significantly elevated levels in CD epithelium, including stem cell populations.
• RNAscope confirmed NLRC5+ epithelial cells colocalising with CD8+ T cells, suggesting epithelial antigen presentation.
• Organoids generated from both NLRC5-deficient and wild type mice and exposed to IFNγ, showed, by flow cytometry, that epithelial cells could present antigen via MHC-I and activate CD8+ T cells. A diminished response was seen in the organoids from NLRC-deficient mice.
• In vivo, NLRC5-deficient mice were partially protected from acute DSS-induced colitis, with reduced epithelial MHC-I, inflammation, and immune cell infiltration.
• Evaluation of whole intestinal mucosal biopsies across multiple patient cohorts found significantly elevated MHC-I and NLRC5 expression, which correlated with disease phenotype and outcomes.
• Machine learning approaches generated a prognostic epigenetic signature linked to clinical severity.

Conclusion

This study identified stable epigenetic dysregulation of intestinal epithelial MHC-I as a novel mechanism in Crohn’s disease. Loss of DNA methylation at NLRC5 and other related genes enhanced epithelial antigen presentation and promoted inflammatory crosstalk with CD8+ T cells, contributing to mucosal pathology. The epigenetic changes were found in intestinal stem cells and were therefore seen in subsequent cell and tissue generations in the absence of an inflammatory environment. These findings reframe the intestinal epithelium as a non-classical antigen-presenting cell in CD, with potential applications for biomarker development and therapeutic targeting. Dennison et al’s research also highlighted the powerful potential of the in vitro organoid platform and its potential uses in disease modelling and translational research.

EPISTEM SERVICES

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Epistem can offer more than 25 years of experience in gastrointestinal stem cell biology, apoptosis and IBD, including 15 years running intestinal organoid models. Our preclinical models are supported by GCLP-accredited PGx and histology laboratories and a range of analytical services including NGS, HLA-typing, bioinformatics, RNAScope, flow cytometry, multiplex cytokine analysis and live cell imaging capabilities. We are used to working a wide range of clients from large pharma to academia on a variety of studies including clinical trials sample analysis, IND-enabling efficacy studies, POC studies and in vitro GI toxicity screening.

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Epistem offer a range of models that allow researchers to look at the importance of different cell types for the development and maintenance of inflammatory bowel disease.  Models of both acute and chronic colitis are available. Key cell populations can be identified by immuno-labelling techniques and can be administered to control disease, or isolated for phenotyping and/or short-term culture and functional assays.A range of analytical platforms are available to support this work, including histology and quantitative image analysis, flow cytometry, luminex-based multiplex analysis of cytokines and chemokines andgene expression analysis

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Epistem offers 3D organoid models from several species that model the intestinal epithelium and allow insights into basic biology, disease and effects of experimental compounds. Multiple assessments can be performed including live imaging, flow cytometry, histology/IHC, TEER (on organoid-derived monolayer cultures) and gene expression.