P0132 ACSS2 inhibition alleviates inflammatory signaling and improves epithelial integrity in Inflammatory Bowel Disease in vitro models
Authors: Mezher N , El-Kurjieh A , Sobh R , Matar M , Nassr S , Kobeissy P H 
Published: January 2025

Abstract

Background
Inflammatory bowel disease (IBD), encompassing Ulcerative Colitis (UC) and Crohn’s Disease (CD), is marked by chronic gastrointestinal inflammation stemming from genetic, environmental, immunological, and microbiota factors1. Acetyl-CoA Synthetase Short Chain Family Member 2 (ACSS2) converts acetate into acetyl-CoA and is primarily cytosolic, but translocates to the nucleus under metabolic stress, where it regulates gene transcription and cellular processes2. Although the gastrointestinal microbiota influences acetate levels, the role of ACSS2 in colitis and intestinal inflammation remains poorly understood3.

Methods
To evaluate ACSS2's role in IBD, we employed in vitro IBD models using Caco-2 and HT-29 intestinal epithelial cell lines. ACSS2 activity was inhibited with an ACSS2 inhibitor, followed by inflammatory stimulation with 100 ng/ml Tumor Necrosis Factor-α (TNF-α) for 30 minutes or 1% Dextran Sodium Sulfate (DSS) for 6 hours. Protein expression levels were assessed via Western blot and immunohistochemistry (IHC) on paraffin-embedded samples from UC patients. The effects of ACSS2 inhibition on inflammatory signaling pathways and cell integrity were assessed using RT-qPCR, immunoblotting, and transepithelial electrical resistance (TEER) measurements.

Results
Treatment of Caco-2 and HT-29 cells with DSS or TNF-α induced upregulation of ACSS2 protein levels compared to controls. IHC analysis of 30 paired paraffin-embedded colon samples from UC patients revealed higher ACSS2 expression and nuclear localization in inflamed sections compared to non-inflamed regions. Inhibition of ACSS2 activity in stimulated Caco-2 and HT29 cells led to a significant decrease in the gene expression levels of pro-inflammatory cytokines such as Interleukin 8 (IL-8), IL-1β and TNF-α. Additionally, immunoblotting demonstrated reduced phosphorylation of p65 and ERK/MEK, indicating suppression of NF-κB and MAPK inflammatory signaling pathways. To assess the effect of ACSS2 inhibition on cell integrity, TEER assays revealed that ACSS2 inhibition preserved cell integrity in DSS-treated monolayers, accompanied by increased expression of tight junction proteins Occludin and ZO-1, suggesting improved epithelial integrity in the inflammatory context.

Conclusion
Our findings demonstrate a pro-inflammatory role for ACSS2 in IBD, mediated through its involvement in inflammatory signaling and epithelial integrity. These results highlight ACSS2 as a potential therapeutic target for IBD and warrant further investigations in more advanced models, including human-derived intestinal organoids and in vivo systems, to validate its role in disease pathogenesis and therapeutic potential.

References
1.Sairenji, T., Collins, K. L. & Evans, D. V. An Update on Inflammatory Bowel Disease. Prim. Care Clin. Off. Pract. 44, 673–692 (2017).
2.Ling, R. et al. Acetyl-CoA synthetase 2(ACSS2): a review with a focus on metabolism and tumor development. Discov. Oncol. 13, 58 (2022).
3.Hao, F. et al. Butyrate enhances CPT1A activity to promote fatty acid oxidation and iTreg differentiation. Proc. Natl. Acad. Sci. 118, e2014681118 (2021).