Abstract
Background and aims Inflammatory Bowel Diseases (IBD) are chronic inflammatory conditions influenced heavily by environmental factors. DNA methylation is a form of epigenetic regulation linking environmental stimuli to gene expression changes and inflammation. Here, we investigated how DNA methylation of the TNF promoter differs between inflamed and uninflamed mucosa of IBD patients, including anti-TNF responders and non-responders.
Methods We obtained mucosal biopsies from 200 participants (133 IBD and 67 controls) and analyzed TNF promoter methylation using bisulfite sequencing, comparing inflamed with uninflamed segments, in addition to paired inflamed/uninflamed samples from individual patients. We conducted similar analyses on purified intestinal epithelial cells from bowel resections. We also compared TNF methylation levels of inflamed and uninflamed mucosa from a separate cohort of 15 anti-TNF responders and 17 non-responders. Finally, we sequenced DNA methyltransferase genes to identify rare variants in IBD patients and functionally tested them using rescue experiments in a zebrafish genetic model of DNA methylation deficiency.
Results TNF promoter methylation levels were decreased in inflamed mucosa of IBD patients and correlated with disease severity. Isolated IECs from inflamed tissue showed proportional decreases in TNF methylation. Anti-TNF non-responders showed lower levels of TNF methylation than responders in uninflamed mucosa. Our sequencing analysis revealed two missense variants in DNMT1, one of which had reduced function in vivo.
Conclusions Our study reveals an association of TNF promoter hypomethylation with mucosal inflammation, suggesting that IBD patients may be particularly sensitive to inflammatory environmental insults affecting DNA methylation. Together, our analyses indicate that TNF promoter methylation analysis may aid in the characterization of IBD status and evaluation of anti-TNF therapy response.
Competing Interest Statement
The authors have declared no competing interest.
Funding Statement
This work was supported by NIH NIDDK grants DK113123 and DK132120 as well as a Kenneth Rainin Foundation Innovator Award to MB. This study was also supported by NIDDK grants P01DK094779, 1R01DK104828, P30-DK034987 and the Helmsley Charitable Trust to SZS. DSL was supported by Duke Training Grant in Digestive Diseases and Nutrition Grant DK007568. LM was supported by NIH NRSA grant DK098885. Research reported in this publication utilized the Biorepository and Molecular Pathology Shared Resource at Huntsman Cancer Institute at the University of Utah and was supported by the National Cancer Institute of the National Institutes of Health under Award Number P30CA042014. EED is the Ann Marie and Francis Klocke, MD Research Scholar.
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Duke University Health System Institutional Review Board gave ethical approval for this work.
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Footnotes
Grant support
This work was supported by NIH NIDDK grants DK113123 and DK132120 as well as a Kenneth Rainin Foundation Innovator Award to MB. This study was also supported by NIDDK grants P01DK094779, 1R01DK104828, P30-DK034987 and the Helmsley Charitable Trust to SZS. DSL was supported by Duke Training Grant in Digestive Diseases and Nutrition Grant DK007568. LM was supported by NIH NRSA grant DK098885. Research reported in this publication utilized the Biorepository and Molecular Pathology Shared Resource at Huntsman Cancer Institute at the University of Utah and was supported by the National Cancer Institute of the National Institutes of Health under Award Number P30CA042014. EED is the Ann Marie and Francis Klocke, MD Research Scholar
Disclosures
The authors have no conflicts of interest to disclose.
Data transparency statement
Data analyzed for this study will be made available upon reasonable request to the corresponding authors.
Synopsis
We analyzed TNF methylation among inflamed and uninflamed mucosae of inflammatory bowel disease patients. TNF hypomethylation was prominent within inflamed tissue, and similar changes were observed in isolated epithelial cells. Anti-TNF non-responders exhibited hypomethylation in both uninflamed and inflamed mucosae.
Data Availability
All data produced in the present study are available upon reasonable request to the authors.
Abbreviations
- BAC
- Bacterial Artificial Chromosome
- CD
- Crohn’s Disease
- CpG
- Cytosine-phosphate-Guanine
- cRNA
- copy RNA
- CRP
- C-Reactive Protein
- DNMT1/3A/3B
- DNA Methyltransferase 1/3A/3B
- ESR
- Erythrocyte Sedimentation Rate
- FACS
- Fluorescence Activated Cell Sorting
- GFP
- Green Fluorescent Protein
- IBD
- Inflammatory Bowel Disease
- IECs
- Intestinal Epithelial Cells
- NIBD
- non-IBD
- TNF
- Tumor Necrosis Factor
- UC
- Ulcerative Colitis
- UHRF1
- Ubiquitin like with PHD and Ring Finger Domains 1