Background: More than one-third of Crohn's disease (CD) patients develop a fibrostenotic phenotype that can ultimately result in intestinal obstruction. The current available anti-inflammatory therapies are not sufficient to revert or treat intestinal fibrosis. Timely intervention focused on reducing fibrosis is still an unmet need in CD. Continuous activation by microbial-associated molecular signatures and/or cytokines may lead to altered gene expression and modulation of signaling pathways via epigenetic mechanisms. These fibroblasts are then able to independently maintain inflammation and initiate the fibrotic process.
This study aimed to elucidate the DNA methylome of fibroblasts cultured from ileal resections from control patients (HC) and non-inflamed (CDNINF), inflamed (CDINF) or stenotic (CDSTE) ileum from CD patients.
Methods: Fibroblasts were acquired and cultured from patients with CD and colon cancer whereby ileal samples were taken at least 10 cm away from cancerous tissue. Genomic DNA from 8 CD patients and 3 HCs was isolated, bisulfite treated and analyzed using the Illumina Human Methylation EPIC BeadChip array. Similarly, RNA from 11 CD patients and 4 HCs was isolated and mRNA expression profiles were obtained using RNA-seq (Illumina NextSeq 500).
Results: First it was established that there were no major differences in the global DNA methylation between cell culture passages. Then, the DNA methylation of fibroblasts from CD was compared to HC and between the various degrees of CD ileum. Approximately 107 reads were obtained for each sample, alignment and mapping was performed using STAR and differential expression analysis was performed using DESeq2. In summary, we found 8, 65,383 and 109,494 significantly differentially methylated positions (DMPs; Benjamini-Hochberg adjusted p-value <0.05) when comparing CD versus HC, CDINF versus CDNINF and CDSTE versus CDNINF respectively. The same trend distribution was found in the transcriptome of these cells. Preliminary RNA-Seq data revealed 4, 9 and 101 differentially expressed genes respectively. Our results show that most differences of the methylome and transcriptome are identified when comparing stenotic versus non-inflamed CD tissue. Preliminary enrichment analyses of the DMPs found in the CDSTE versus CDNINF comparison suggested an association to processes such as cellular differentiation and growth, which corroborates the excessive growth phenotype typical of stenosis.
Conclusions: Our study revealed that the DNA methylome and transcriptome of fibroblasts show several differences in CD patients versus control patients and that among the different degrees of CD most differences are observed when comparing stenotic with non-inflamed tissue.