Background: Recurrent cycles of intestinal inflammation and repair drive fibrosis and stricture formation in patients with Crohn's disease (CD). Histone acetylation is an important epigenetic mechanism that impacts gene transcription and is controlled by the histone deacetylase enzymes (HDACs). This pathway can be pharmacologically manipulated by HDAC inhibitors such as valproic acid (VPA), which protects against experimentally induced colitis and reduces fibrosis in non-intestinal models of disease. In this study we investigate the relationship between histone-3 acetylation levels and inflammation and fibrosis in CD and test the ability of VPA to modulate synthesis of collagen I.
Methods: Histone-3 acetylation was assessed by immunohistochemistry in FFPE tissue from healthy controls (n=12), CD patients with: quiescent (n=8); inflammatory (n=7); and fibrotic disease (n=6). The effects of VPA (5mM) on collagen I synthesis, measured by qPCR, immunofluorescence and ELISA, were tested in ex-vivo CD gut cultures, a novel 3D organotypic model of the gut and a human intestinal fibroblasts cells line (CCD18co cells). An Illumina HT12 array was also performed on RNA extracted from CCD18c0 cells (n=4) to further elucidate pathways regulated by VPA. The array was analysed using “significance analysis of microarray” package in R, and pathway analysis performed using the Reactome database.
Results: Relative to healthy controls and CD patients with quiescent disease, there was a reduction in histone-3 acetylation in the mucosa overlying both inflamed and strictured gut (p=0.004, ANOVA). Histone-3 acetylation was also lower in strictured gut relative to adjacent non-strictured areas (p=0.015, n=6). Ex-vivo cultures of CD biopsies with VPA, which increases histone-3 acetylation, led to a reduction in collagen I RNA expression (COL1A2, p=0.005, n=8). Levels of collagen I protein were also lower in conditioned media taken from an organotypic model of the gut treated with VPA (p=0.016, n=3). Gene expression arrays further identified genes enriched in 24 pathways that were regulated by VPA, including the collagen degradation pathway (p=0.044), with a reduction in collagen I protein confirmed by immunofluorescence (p=0.013, n=4).
Conclusions: Hypoacetylation is a pathological feature of both inflammation and fibrosis in CD. Treatment with VPA increases histone acetylation and suppresses collagen expression in CD patient biopsies and models of intestinal fibrosis, demonstrating a direct link between histone acetylation and collagen production in CD. The data highlight the potential to exploit existing drugs that modulate histone acetylation in the treatment of fibrostenosing CD.