DOP034 Intestinal stem cell dysfunction is driven by loss of HMGCS2-mediated ketogenic response in Ulcerative colitis and represents a novel therapeutic target
Authors: Rutherford D , Whelan R , Lau P , Hodge M , Cholewa-Waclaw J , Adams C , Cavanagh A , Greaves L , Smith A , Jones G , Ho G T 
Published: January 2025

Abstract

Background
Intestinal stem cells (ISCs) continuously divide and differentiate into multiple lineages of enterocytes, a process that places high energy demands. Recent evidence implicates bioenergetic failure of ISCs with non-resolving inflammation in Ulcerative Colitis (UC)1.

Methods
In newly diagnosed, treatment-naïve UC patients, we characterized mitochondrial oxidative phosphorylation (OXPHOS) and metabolic proteins in epithelial and ISCs, performed single-cell RNA sequencing (scRNA-seq) to identify deregulated metabolic pathways, and assessed their biological relevance on intestinal regeneration with in-depth ex vivo metabolic platforms, including SCENITH (Single Cell Energetic Metabolism by Profiling Translation Inhibition).

Results
Single-cell quantitative immunofluorescence showed loss of mitochondrial OXPHOS Complex I and IV in UC (n=10/group; p&lt;0.0001) and UC-derived organoids (n=20/group; p&lt;0.0001), suggesting bioenergetic failure transmitted from ISCs to daughter epithelial cells. scRNA-seq of ISCs from active UC revealed significant ketogenesis pathway, downregulation, with HMGCS2 as the top downregulated gene (FC -2.76, p&lt;0.0001). HMGCS2, the rate-limiting mitochondrial matrix ketogenesis enzyme, produces the ketone body βHB which, alongside providing metabolites for the TCA cycle, has important roles in stem cell fate-signalling2 (Figure 1). HGMCS2 expression was reduced along the crypt axis in active UC (n=20/group; p&lt;0.001). Moreover, we find via colorimetric assay that colonic organoids produce and secrete βHB, and this is reduced in active UC (n= 5/group, p &lt;0.001). βHB supplementation of UC organoids restores ISC function, measured via growth assay (n=5/group, p&lt;0.05, Figure 2). Further scRNA-seq of βHB treated UC organoids revealed that βHB aids mitochondrial function and reduces cellular stress in ISCs, lowering expression of genes associated with stress and protein misfolding (HSPD1 and VDAC3), while increasing mitophagy (PINK1). Functionally, we confirm this by showing βHB restored OXPHOS capacity in UC organoids using SCENITH (n=3/group, p&lt;0.05). βHB also reduced cellular and mitochondrial reactive oxygen species, measured via Cellrox (n=9/group, p&lt;0.01). In UC organoids, fenofibrate (PPARα agonist), induced HMGCS2 expression (p&lt;0.001) and βHB production (p&lt;0.05). Finally, HMGCS2 protein expression significantly increased in responders to therapy with mucosal healing (n=4 paired samples, p&lt;0.05).

Conclusion
We provide the first combined evidence of mitochondrial dysfunction and ketogenesis failure (‘primary’ and ‘backup’ energy failure) in UC. Crucially, restoring ketogenesis reverses the ISC pathogenic defect thus, opening a new ‘immuno-metabolic’ therapeutic approach in UC.

References
1. Ho G, Theiss AL. Mitochondria and Inflammatory Bowel Diseases: Toward a Stratified Therapeutic Intervention. Annu Rev Physiol. 2022;84(1). doi:10.1146/annurev-physiol-060821-083306
2. Cheng CW, Biton M, Haber AL, et al. Ketone Body Signaling Mediates Intestinal Stem Cell Homeostasis and Adaptation to Diet. Cell. 2019;178(5):1115-1131.e15. doi:10.1016/j.cell.2019.07.048