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P063 HDAC as versatile regulators of the intestinal epithelial barrier in inflammatory bowel disease

Friedrich M., Ganther J., Breiderhoff T., Rosenthal R., Glauben R., Siegmund B.

Charité - Universitätsmedizin Berlin, Medical department for Gastroenterology, Infectious Diseases and Rheumatology, Berlin, Germany

Background

Recent studies have highlighted the importance of the intestinal mucosal barrier function in maintaining gut homeostasis as well as contributing to disease pathophysiology of inflammatory bowel disease (IBD). When we could demonstrate that pan-inhibition of histone deacetylases (HDAC) ameliorates experimental colitis and colitis associated tumorigenesis, HDAC moved into our focus as promising targets for diagnosis and therapy. Even though a specific immune regulatory function of various single HDAC have already been shown, the impact of single HDAC on the intestinal epithelial barrier is little understood.

Here, we aim to investigate the role of HDAC on the epithelial barrier and further assess the impact of HDAC7 on barrier function by analyzing the role of HDAC7 in the physiology of colonic epithelial cells (CEC) under pro-inflammatory conditions with special emphasis to IBD and CRC.

Methods

To define the relevance of HDAC for gut homeostasis primary CEC were isolated from human biopsies and analyzed for HDAC expression using quantitative PCR. Further we examine effects of pan-HDAC inhibitors on the integrity of the gut epithelial barrier by treating human T84 and murine CMT93 cells with Givinostat and Vorinostat. For functional characterization of HDAC7, KO cells were generated from murine CMT93 cells by using the CRISPR/Cas9 system. Cell migration was assessed via wound healing assay. Integrity and functionality of the monolayer were measured by the trans-epithelial electrical resistance (TER), cytokine secretion and the trans-epithelial flux of 4 kDa FITC-Dextran.

Results

Expression analysis revealed an overall reduction of HDAC in IBD (Crohn's disease and Ulcerative colitis). Human T84 and murine CMT93 cells demonstrated enhanced cell migration, increased secretion of regenerating IL-8 as well as a reduced decrease of the TER under inflammatory conditions when treated with HDAC inhibitor. An enhanced integrity and functionality of the monolayer could also be confirmed by an impaired flux of 4 kDa FITC-Dextran in the presence of HDAC inhibitor. On the other hand functional analysis of single HDAC KO cells displayed divergent effects as shown by a reduced TER as well as delayed cell migration in CRISPR/Cas9 generated HDAC7 KO cells. These effects were accompanied by a reduced expression of cell adhesion and migration molecules as shown by RNA sequencing.

Conclusion

Our results indicate an integrated role of HDAC in the maintenance of the intestinal barrier and further point to a regulatory function of HDAC7 in the development of the intestinal epithelial barrier and the inflammatory response of CEC.